Oncogene, recombinant protein derived therefrom, and uses thereof

ABSTRACT

The present invention identifies the total nucleotide sequence of a novel oncogene from human, which is directly involved in such a cancerization mechanism as for cervical cancer induced by HPV infection of cervical epithelial cell and the amino acid sequence of an oncogenic protein encoded thereby, and to provide a full-length polynucleotide encoding a peptide chain of the oncogenic protein derived from the novel oncogene, which can be used for recombinant production of the oncogenic protein, and the peptide chain of the oncogenic protein produced recombinantly therewith. Specifically, the present invention provides a novel oncogene polynucleotide from human involving development of cervical cancer, comprising a nucleotide sequence encoding an amino acid sequence of SEQ. ID. No.1, particularly a polynucleotide of the nucleotide sequence of SEQ. ID. No.2.

FIELD OF THE INVENTION

The present invention relates to a novel oncogene from human, which isinvolved in development of human cervical cancer, a recombinant proteinderived from the oncogene and uses thereof in medical applications.

BACKGROUND ART

There are many documents reporting that chromosome instability isinvolved in development of a cancer. In addition, it has been recentlydemonstrated that a defect in a molecule controlling a checkpoint duringthe G2/M phase in a cell cycle causes chromosome instability. However,since in many carcinoma cells, a gene defect in a molecule controlling acheckpoint in the cell is not frequently observed, a mechanism ofinducing chromosome instability, which is substantially involved inonset of the cancer, remains still unclear in many aspects.

It is widely known that development of cervical cancer involvesinfection with a human papilloma virus (HPV) such as types of HPV-16 orHPV-18. In a cervical cancer tissue, HPV infection has been observed ata frequency of 90% or more. In a development mechanism of cervicalcancer induced by HPV infection, E6 and E7 gene products of the virusplay an important role. Specifically, it is known that E6 accelerates aprocess for digesting p53 tumor suppressor protein, while E7 blockscanceration-inhibiting activity of pRB (retinoblastoma) tumor suppressorprotein that is a Rb gene product, which two steps result intumorigensis. A specific oncogenic protein activated by HPV infectionhas not, however, been identified yet. Particularly, E6 and E7, viralgene products of HPV, induce chromosome instability and carcerize acell, but detail of its mechanism directly related thereto is leftsubstantially unknown in variety of aspects. For approaching totreatment of cervical cancer, it is, therefore, very important toidentify an oncogenic protein as a target for HPV and an encodingoncogene thereof. Cervical cancer progresses from a precancerous state,i.e., dysplasia (epithelial dysplasia of cervical squamous cell) toinvasive cancer. Depending on a case, the disease may remain in thedysplasia stage without progressing to cancer. On the other hand, thereare considerable cases where dysplasia may rapidly progress to anadvanced cancer. In view of the situation, it may be important for moreaccurate cancer diagnosis to identify a molecule directly involved indevelopment of cervical cancer.

DISCLOSURE OF INVENTION

As described above, in the process where HPV infection of cervicalepithelial cell induces development of an invasive cancer via aprecancerous state, dysplasia, its direct origin would be considered tobe a mechanism where an expression-inhibiting activity of p53 tumorsuppressor protein or pRB tumor suppressor protein, which has inhibitedexpression of some oncogene, is damaged, and the damage leads theoncogene to a high-level expression state. Therefore, the fullnucleotide sequence of the oncogene and an oncogenic protein encodedthereby must be first identified, which opens a way for developing meansfor inhibiting the biochemical functions of the oncogenic protein andfurther means for blocking a cancerization mechanism advanced by theoncogenic protein.

Furthermore, identification of the full nucleotide sequence of theoncogene and the amino acid sequence of the oncogenic protein encodedtherein may allow us to produce a nucleic acid probe for detectingexpression of an mRNA transcribed from the oncogene or to generate aspecific antibody to the oncogenic protein with use of a recombinantoncogenic protein thereof. In other words, it may allow us to developdiagnosis means utilizing the nucleic acid probe or specific antibody,which is useful for diagnosing an early step of developing course to aninvasive cancer via a precancerous state, dysplasia, that is caused byHPV infection in uterine cervix.

For solving the above problems, an aim of the present invention is toidentify the full nucleotide sequence of a novel oncogene from Human andthe amino acid sequence of an oncogenic protein encoded therein, whichis directly involved in a cancerization mechanism in, for example,cervical cancer caused by HPV infection into cervical epithelial celland, and also to provide a full-length polynucleotide encoding a peptidechain of the oncogenic protein, that is derived from the novel oncogene,which can be used for recombinant production of the oncogenic protein,as well as the peptide chain of the oncogenic protein recombinantlyproduced therewith.

We have intensely studied for solving the above problems, and finallyhave found and cloned a gene increasing expression in a cervical cancercell when adding an environmental hormone thereto. We have concludedthat the gene cloned is one of oncogenes, because

(1) the gene is highly expressed in a carcinoma cell;

(2) cervical cancer is caused by HPV infection, and expression of E6 andE7 proteins by transducing E6 and E7 genes from HPV to the cell enhancethe expression of said gene;

(3) p53 protein inhibits activity of the promoter region in said gene;

(4) lack or mutation of p53 protein is indeed involved in development ofcervical cancer;

(5) expression of said gene can be inhibited by a double strand ofinterfering short-chain RNA(siRNA) to arrest growth of the cancer, andafter further investigation, have achieved the present invention.

Thus, an oncogene polynucleotide according to the present invention is anovel oncogene polynucleotide derived from human involving developmentof cervical cancer, comprising a nucleotide sequence encoding an aminoacid sequence of SEQ. ID. No.1. In particular, it is the polynucleotide,wherein the nucleotide sequence encoding the amino acid sequence of SEQ.ID. No.1 is a nucleotide sequence of SEQ. ID. No.2.

The present invention also provides an invention of a peptide or itssalts produced recombinantly, based on the above oncogene polynucleotideaccording to the present invention. Specifically, the recombinantpeptide of the present invention recombinant peptide or its salts,comprising the amino acid sequence of SEQ. ID. No.1 or a partial aminoacid sequence of the amino acid sequence. In particular, it may be arecombinant oncogenic protein comprising the amino acid sequence of SEQ.ID. No.1. The present invention also provides a recombinant vectorcomprising a polynucleotide encoding the recombinant peptide, which isusable for preparing said recombinant peptide therewith. For example,when aimed is the recombinant oncogenic protein of the present inventionconsisting of the amino acid sequence of SEQ. ID. No.1, said recombinantvector therefor is a recombinant vector containing an oncogenepolynucleotide comprising a nucleotide sequence encoding the amino acidsequence of SEQ. ID. No.1, particularly containing a polynucleotidewherein the nucleotide sequence encoding the amino acid sequence of SEQ.ID. No.1 is a nucleotide sequence of SEQ. ID. No.2.

The present invention also provides a transformed cell produced bytransforming a host cell using said recombinant vector; for example, atransformed cell produced by transforming a host cell with therecombinant vector targeted to said recombinant oncogenic protein of thepresent invention. Therefore, a process for producing the recombinantpeptide or its salts of the present invention is

a process for producing a recombinant peptide or its salts derived froman oncogene of the present invention comprising the steps of:

culturing the above transformed cell to allow the transformed cell toproduce the recombinant peptide of the present invention; and

collecting the recombinant peptide produced from the culture. Inparticular, it may be preferably a process for producing a recombinantoncogenic protein of the present invention comprising the steps of:

culturing said transformed cell in which the full-length gene DNA hasbeen transformed to allow the transformed cell to produce therecombinant oncogenic protein of the present invention; and

collecting the recombinant oncogenic protein produced from the culture.

With use of the above recombinant peptide of the present invention, thepresent invention provides an invention of an antibody that is aspecific antibody generated using the recombinant peptide as animmunogen. For example, an antibody of the present invention may be anantibody exhibiting a specific reactivity to the partial amino acidsequence of 623 to 1185 region of the amino acid sequence of SEQ. ID.No.1.

Furthermore, the present invention provides an antibody reagent kit foran antigen-antibody reaction comprising said specific antibody,available for detecting an oncogenic protein comprising the amino acidsequence of SEQ. ID. No.1 or a peptide fragment derived from theoncogenic protein. Alternatively, the present invention provides adiagnosis kit being usable for detection of an oncogenic proteincomprising the amino acid sequence of SEQ. ID. No.1 or a peptidefragment derived from the oncogenic protein by means of anantigen-antibody reaction, comprising the specific antibody of thepresent invention.

The present invention also provides an antisense polynucleotidecomprising a complementary nucleotide sequence to a partial nucleotidesequence of the nucleotide sequence of SEQ. ID. No.2, which is a DNAfragment having at least a length selected from the region of 15 to 300bases. In addition, as for a probe hybridization kit according to thepresent invention, it also provides a probe hybridization kit availablefor detecting an mRNA comprising the nucleotide sequence of SEQ. ID.No.2, its partial nucleotide sequence therein or cDNA prepared by themRNA, comprising the above antisense polynucleotide as the DNA probe.Alternatively, the present invention provides also a diagnosis kitavailable for detecting expression of mRNA comprising the nucleotidesequence of SEQ. ID. No.2, which is translated into an oncogenic proteincomprising the amino acid sequence of SEQ. ID. No.1, by means of a probehybridization method, comprising said antisense polynucleotide as thehybridization probe.

On the other hand, the present invention also provides a primer pair forPCR amplification of cDNA comprising the nucleotide sequence of SEQ. ID.No.2, consisting of paired primers of:

a nucleotide sequence 5′-TTGGATCCATGACATCCAGATTTGGGAAAACATACAGTAGG-3′(SEQ ID NO: 3); and

a nucleotide sequence 5′-TTGAATTCCTAGCAATGTTCCAAATATTCAATCACTCTAGA-3′(SEQ ID NO: 4), and also the present invention provides a primer pairfor PCR amplifying a partial chain in cDNA comprising the nucleotidesequence of SEQ. ID. No.2, consisting of paired primers of:

5′-GAATTCATAGGCACAGCGCTGAACTGTGTG-3′ (SEQ ID NO: 5); and

5′-TTGAATTCCTAGCAATGTTCCAAATATTCA-3′ (SEQ ID NO: 6).

Otherwise, as for an invention of a double strand of an short-chaininterfering RNA, the present invention provides a double strand of anshort-chain interfering RNA capable of inhibiting expression of mRNAcomprising the nucleotide sequence of SEQ. ID. No.2 in a cervical cancercell, wherein the double strand of the siRNA has a nucleotide sequence:CGGACTACCCTTAGCACAA (SEQ ID NO: 7). In addition, it may be also appliedto a pharmaceutical composition for inhibiting expression of mRNAcomprising the nucleotide sequence of SEQ. ID. No.2 in a cervical cancercell to arrest growth of the carcinoma cell, comprising said doublestrand of short-chain interfering RNA of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows comparison of an amino acid sequence between a dWAPL ofDrosophia reported and an hWAPL of the present invention.

FIG. 2 shows an alignment of amino acids identical or similar to thosein the dWAPL in a partial amino acid sequence of 623 to 1185 amino acidslying on the C-terminus side for the hWAPL of the present invention.

FIG. 3 shows very high homology between the amino acid sequences of ahuman WAPL protein and a mouse WAPL protein.

FIG. 4 shows the results of Western blotting analysis for extracts fromSaos-2 cell (lanes 2, 3) and NIH3T3 cell (lane 1) by using ananti-hWAPL-N antibody and an anti-hWAPL-C antibody.

FIG. 5 shows (A): Northern blotting for evaluating an expression levelof an hWAPL protein in carcinoma cells (T) of ovarian cancer, pulmonarycancer, colorectal carcinoma, corpus uteri cancer and cervical cancer incomparison with normal cells (N) corresponding thereto; (B): the resultsof real-time PCR for confirming expression of hWAPL gene in carcinomacells (T) of cervical cancer, corpus uteri cancer, ovarian cancer,breast cancer, gastric cancer, renal cancer, colorectal carcinoma normalcells in comparison with (N) corresponding thereto; and (C): the resultsof RT-PCR detection of mRNA expression of E6/E7 gene derived from HPV inthe carcinoma cells (T).

FIG. 6 shows (A): Western blotting for confirming expression of thehWAPL protein, which is induced by E6 and E7 recombinant proteinsderived from HPV 16, and cleavage of the p53 suppressor protein therebyin HDK1 cell; and (B): Western blotting for confirming inhibition oftranscription of E6 and E7 genes, increase in a p53 suppressor proteinlevel and inhibition of expression of the hWAPL protein by BPV-derivedE2.

FIG. 7 shows (A): chromosome instability and increase of polyploid; and(B): increase in a frequency of micronuclei formation induced byover-expression of a GFP-hWAPL fused protein in HeLa cell.

FIG. 8 shows chromosome instability and increase in a frequency ofmultinucleation induced by over-expression of a GFP-hWAPL fused proteinin a HeLa cell.

FIG. 9 shows cancerization induction of NIH 3T3 fibroblast by the hWAPLprotein, specifically (A): formation of a focus structure in culturing aHA-hWAPL 3T3 cell strain; (B): formation of a tumor in a site in a nudemouse, to which the HA-hWAPL 3T3 cell strain has been injected; (C):over-expression of the HA-tagged hWAPL protein in a tumor formingregion; and (D): heterotypic mitosis in a cancerized cell.

FIG. 10 shows the results of evaluating cell growth inhibiting effect byhWAPL siRNA in SiHa cell derived from HPV16 positive cervical cancer,displayed with a plot of the cell number (×10³ in unit) (in ordinate) toa time from transduction of the siRNA (hour in unit) (in abscissa).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in detail hereafter.

We have searched for a human-derived protein which is inducing factorfor chromosome instability, assuming that chromosome instability wouldbe considerably involved in cancerization mechanism in a cervical cancercell. We have searched particularly for a protein having highpotentiality for inducing heteroploidy or isogene formation amongchromosome instability events.

We have noticed that dWAPL protein has been reported as a proteincontrolling a heterochromatin structure during an interkinesis in ameiosis process, among a variety of proteins derived from Drosophilamelanogaster (fruit-fly) whose genomic genes have been most studiedamong animals. Specifically, we have found that when such a functionthat the protein exhibits for controlling the heterochromatin structureis expressed in a mitosis process in a normal cell, heteroploidy orisogene formation may be occasionally induced. We have first studiedwhether a protein corresponding to such a dWAPL is actually encoded on ahuman genomic gene. Based on the nucleotide sequence of the dWAPL genereported (GenBank accession No.U40214), we have searched for fragmentsshowing significant similarity from cDNA fragments registered in GenBankas gene fragments originated from human and have selected the KIAA0261fragment as that comprising a nucleotide sequence similar to the dWAPLgene.

For identifying a full-length cDNA comprising the KIAA0261 fragment, wehave searched in the EST database for a human-derived expression-tagnucleotide sequence, which may be a fragment comprising anun-translation part which is presumably an upstream nucleotide sequencelying in the 5′-side of the KIAA0261 fragment, and have selected ESTclones: BE410177, BF79516 and BE257022.

We have determined an upstream nucleotide sequence lying in the 5′-sideof the KIAA0261 fragment, using a 5′-RACE method with reference to theEST clones. Furthermore, since there is high probability that a proteinhaving a function similar to the dWAPL is actually expressed in a cellhaving a meiosis process, we have cloned a full-length cDNA comprisingthe nucleotide sequence of the KIAA0261 fragment as well as the upstreamnucleotide sequence determined above in the 5′-side thereof, using acommercially available cDNA library, i.e., a human testicular cDNA kit(Marathon-Ready™ cDNA Kit; Clontech Inc.) as a template.

Practical sequencing has indicated that the coding region in the clonedfull-length cDNA has 3570 base pairs, deducing an amino acid sequencewith 1190 amino acids corresponding thereto. It is referred to “humanWAPL (hWAPL)” as a human-derived protein similar to the dWAPL protein,and the corresponding gene is referred to as “hWAPL gene”. Thefull-length nucleotide sequence that is corresponding to the ORF in thehWAPL gene is represented by SEQ. ID. No.2, and the deduced amino acidsequence of the hWAPL protein is represented by SEQ. ID. No.1.Comparison of the amino acid sequence encoded by the ORF of the hWAPLgene of the present invention, i.e., the deduced amino acid sequence ofthe hWAPL protein, with the amino acid sequence of the dWAPL proteinindicates, as shown in FIG. 1, that there is an identity of 35% and asimilarity of 53% for a partial sequence of amino acids 623 to 1185lying in region of the C-terminus. The amino acids exhibiting such anidentity and a similarity are shown in FIG. 2.

In addition, we have cloned a cDNA encoding a mouse-derived homologuethereto, a mouse WAPL protein, assuming that some mammals other thanhuman may have also a corresponding protein. After sequencing it, theamino acid sequences encoded therein were compared. In practice, it hasbeen confirmed that the human WAPL protein and the mouse WAPL proteinshows very high homology to each other. FIG. 3 shows the result ofcomparative alignment. We have registered the full-length nucleotidesequence for coding region in the human WAPL gene under an accession No.AB065003 in DDBJ/EMBL/GenBank.

We have conducted Western blotting analysis for extracts from Saos-2cell and NIH3T3 cell, using an anti-hWAPL-N antibody and an anti-hWAPL-Cantibody, which are antibodies specific to the peptide chain in thehuman WAPL protein, as explained in Example 6. As shown in FIG. 4, theresults have revealed a band of a protein reactive to the antibody withapproximately 140 kDa. Thus, it has been confirmed that the human WAPLprotein is actually present in the human-derived cell in some extent.

Furthermore, based on the following various confirmation methods:

EXAMPLE 2

expression of the hWAPL gene in a human cancer tissue;

EXAMPLE 3

induction of expression of the hWAPL gene by E6 and E7 derived from HPVtype 16;

EXAMPLE 4

inhibition of promoter activity of the hWAPL gene product by the p53suppressor protein;

EXAMPLE 7

induction of chromosome instability by the hWAPL protein;

EXAMPLE 8

induction of cancerization of NIH 3T3 fibroblast by the hWAPL protein,it can be concluded that the hWAPL gene is an oncogene at least involvedin a mechanism of development of cervical cancer.

The plasmid pGEMhWAPL comprising the full-length cDNA of the hWAPL gene,which is used in producing the transformant containing the full-lengthcDNA of the WAPL gene; Escherichia coli DH5 pGEMhWAPL strain that wasobtained in Example 1, as described below, have been deposited on Jan.7, 2003 as an original deposition date, in International Patent OrganismDepositary (National Institute of Bioscience and Human-Technology) inNational Institute of Advanced Industrial Science and Technology at Chuo6^(th), 1-1-1, Higashi, Tsukuga, Ibaragi, 305-8566, under a depositnumber of FERM BP-8269 in the International Depositary Authority underthe Budapest Treaty.

We have also confirmed that expression of the hWAPL protein as a hWAPLgene product is inhibited by using a double strand of short-chaininterfering RNA (siRNA). Specifically, a double strand of short-chaininterfering RNA (siRNA) exhibiting such expression inhibition activitymay include, for example, that having a nucleotide sequence:CGGACTACCCTTAGCACAA (SEQ ID NO: 7). In such a case, further growth ofthe carcinoma cell is also inhibited, so that development of the cancercan be arrested.

As for a protein having an amino acid sequence identical orsubstantially identical to the amino acid sequence of SEQ. ID. No.1 ofthe present invention (hereinafter, sometimes referred to as “a proteinof the present invention”), examples thereof may include an amino acidsequence having a homology of about 70% or more, preferably about 80% ormore, more preferably 90% or more, most preferably about 95% or morewith the amino acid sequence of SEQ. ID. No.1.

As for the protein comprising an amino acid sequence identical orsubstantially identical to the amino acid sequence of the SEQ. ID. No.1, preferred may be such a peptide that has an amino acid sequencesubstantially identical to the amino acid sequence of SEQ. ID. No. 1 andpossesses a substantially equivalent activity to the protein having theamino acid sequence of SEQ. ID. No.1.

The substantially equivalent activity may include, for instance,activity of the protein comprising the amino acid sequence of SEQ. ID.No. 1, such as a function for inducing cancer, enzymic activity,transcribing activity and binding activity with a binding proteintherefor.

The term “substantially equivalent” as used herein means that theseactivities are identical in nature (for example, biochemically orpharmacologically).

Examples of the amino acid sequence being identical or substantiallyidentical to the amino acid sequence of SEQ. ID. No.1 include

(i) the amino acid sequence of SEQ. ID. No.1;

(ii) an amino acid sequence which has deletions of 1 to 30, preferably 1to 20, more preferably 1 to 10 amino acids from the amino acid sequenceof SEQ. ID. No.1;

(iii) an amino acid sequence which has addition of 1 to 30, preferably 1to 20, more preferably 1 to 10 amino acids to the amino acid sequence ofSEQ. ID. No.1;

(iv) an amino acid sequence which has insertion of 1 to 30, preferably 1to 20, more preferably 1 to 10 amino acids into the amino acid sequenceof SEQ. ID. No.1;

(v) an amino acid sequence which has replacements of 1 to 30, preferably1 to 20, more preferably 1 to 10 amino acids in the amino acid sequenceof SEQ. ID. No.1 with other amino acids; and

(vi) an amino acid sequence which has combinational modification of twoor more selected from the above (ii) to (v)

An example of a protein of the present invention may be a proteincomprising the amino acid sequence of SEQ. ID. No.1.

A partial peptide of the present invention may be any of partialpeptides of the protein of the present invention described above, andgenerally preferred is a peptide that is consisted of at least 5 ormore, preferably at least 10 or more amino acids, and further preferablyhas activity equivalent to a protein of the present invention.

In a protein of the present invention or its partial peptide(hereinafter, sometimes referred to as “proteins of the presentinvention”), the left end is N-terminus (amino terminus) while the rightend is C-terminus (carboxy terminus) according to conventional notationfor peptide.

In a protein of the present invention including a protein comprisingamino acid sequence of the SEQ. ID. No.1, C-terminal may be selectedfrom carboxy (—COOH), carboxylate (—COO⁻), amide (—CONH₂) and ester(—COOR).

Examples of R for the ester include C₁₋₆-alkyls such as methyl, ethyl,n-propyl, isopropyl and n-butyl; C₃₋₈-cycloalkyls such as cyclopentyland cyclohexyl; C₆₋₁₂-aryls such as phenyl and α-naphthyl;phenyl-C₁₋₂-alkyls such as benzyl and phenethyl; C₇₋₁₄-aralkyls such asα-naphthyl-C₁₋₂-alkyls including α-naphthylmethyl; and pivaloyloxymethylcommonly used as ester for oral application.

When a protein of the present invention has a carboxy group (orcarboxylate) in a position other than C-terminus, a protein in which thecarboxy group is amidated or esterified is also a protein of the presentinvention. The ester here may be the C-terminal ester described above.

Furthermore, examples of a protein of the present invention include aprotein in which an amino group in an N-terminal amino acid residue (forexample, methionine residue) is protected by a protective group such asC₁₋₆-acyl including C₁₋₆-alkanoyl such as formyl and acetyl; a proteinin which N-terminal glutamic acid residue formed by in vivo cleavage isconverted into form of pyroglutamic acid; a protein in which asubstituent on a side chain in an amino acid residue in the molecule(for example, —OH, —SH, amino, imidazole, indole and guanidino) isprotected by an appropriate protective group (for example, such asC₁₋₆-acyls including typically C₁₋₆-alkanoyl such as formyl and acetyl);and a conjugated protein such as a so-called glucoprotein in which asugar chain is linked.

A salt of a protein of the present invention may be a salt with aphysiologically acceptable acid (for example, inorganic and organicacids) or base (for example, an alkali metal salt), particularlypreferably a physiologically acceptable acid-addition salt. Examples ofsuch a salt include salts with inorganic acids such as hydrochloricacid, phosphoric acid, hydrobromic acid and sulfuric acid, and withorganic acids such as acetic acid, formic acid, propionic acid, fumaricacid, maleic acid, succinic acid, tartaric acid, citric acid, malicacid, oxalic acid, benzoic acid, methanesulfonic acid andbenzenesulfonic acid. Hereinafter, such a salt is also included in aprotein of the present invention.

A protein of the present invention or its salt may be prepared by aknown process for purification of a protein from human or warm-bloodedmammalian cells, or alternatively by culturing a transformant producedby transformation with a DNA encoding the protein described below.

When producing from a human or mammal tissue or cell, the human ormammal tissue or cell is homogenized and extracted with an acid. Theextract is then purified by combined chromatographical procedures suchas reverse phase chromatography and ion-exchange chromatography toisolate a desired product.

A polynucleotide encoding an oncogenic protein of the present inventionmay be any polynucleotide comprising the above nucleotide sequenceencoding an oncogenic protein of the present invention (DNA or RNA,preferably DNA). The polynucleotide may be a DNA or RNA such as an mRNAencoding an oncogenic protein of the present invention, which may besingle or double stranded. When being double stranded, it may be adouble-stranded DNA, a double-stranded RNA or a hybrid of DNA:RNA. Whenbeing single stranded, it may be a sense chain, i.e., a coding chain, oran antisense chain, i.e., a non-coding chain.

A polynucleotide encoding a protein of the present invention may be usedto quantify an mRNA of a protein of the present invention, according toa known method or its modification, for example, a method described inExperimental Medicine Extra Edition “Novel PCRs and their applications”,15(7), 1997.

A DNA encoding a protein of the present invention may be any DNAcomprising the above nucleotide sequence encoding a protein of thepresent invention, and may be a genome DNA, a genome DNA library, theabove cell/tissue-derived cDNA, the above cell/tissue-derived cDNAlibrary or a synthetic DNA.

A vector used for a library may be a bacteriophage, plasmid, cosmid orphagemid. A preparation of a total RNA or mRNA fraction from the cell ortissue may be used for amplification by a direct Reverse TranscriptasePolymerase Chain Reaction (hereinafter, referred to as “RT-PCR”).

A nucleotide sequence available for a probe DNA of the present inventionmay be any sequence such as a DNA sequence which comprises a nucleotidesequence hybridizable with the nucleotide sequence of SEQ. ID. No.2under high stringent conditions and encodes a protein having an activitysubstantially equivalent to that of a protein comprising the amino acidsequence of SEQ. ID. No.1.

A nucleotide sequence hybridizable with the nucleotide sequence of SEQ.ID. No.2 under high stringent conditions may be a nucleotide sequencehaving a homology of about 70% or more, preferably about 80% or more,more preferably about 90% or more, further preferably about 95% or morewith the nucleotide sequence of SEQ. ID. No.2.

Hybridization may be conducted in accordance with a known method or itsmodification, for example, a method described in Molecular Cloning 2nd(J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When using acommercially available library, hybridization can be conducted inaccordance with an attached manual. More preferably, it can be conductedunder high stringent conditions.

High stringent conditions may include, for example, a sodiumconcentration of about 19 to 40 mM, preferably about 19 to 20 mM and atemperature of about 50 to 70° C., preferably about 60 to 65° C., mostpreferably a sodium concentration of about 19 mM and a temperature ofabout 65° C.

More specifically, a DNA encoding a protein comprising amino acidsequence of the SEQ. ID. No.1 may be a DNA comprising the nucleotidesequence of SEQ. ID. No.2.

A DNA encoding a partial peptide of the present invention may be any DNAcomprising a nucleotide sequence encoding the above partial peptide ofthe present invention, and may be a genome DNA, a genome DNA library,the above cell/tissue-derived cDNA, the above cell/tissue-derived cDNAlibrary or a synthetic DNA.

A DNA encoding a partial peptide of the present invention may be a DNAcomprising a partial nucleotide sequence of a DNA comprising thenucleotide sequence of SEQ. ID. No.2, or a DNA comprising a partialnucleotide sequence of a DNA which comprises a nucleotide sequencehybridizable with the nucleotide sequence of SEQ. ID. No.2 under highstringent conditions and encodes a protein having an activitysubstantially equivalent to that of a protein comprising the amino acidsequence of SEQ. ID. No.1.

The nucleotide sequence hybridizable with the nucleotide sequence ofSEQ. ID. No.2 is defined as described above.

A hybridization method and the high stringent conditions may be asdescribed above.

A polynucleotide comprising a part of a DNA sequence encoding a proteinof the present invention or its partial peptide (hereinafter, sometimesreferred to as “a protein of the present invention”) or a part of anucleotide sequence complementary to the DNA may encompass a DNA as wellas an RNA encoding a protein of the present invention or its partialpeptide.

According to the present invention, an antisense polynucleotide (nucleicacid) capable of inhibiting replication or expression of a protein geneof the present invention designed and synthesized on the basis ofnucleotide sequence data on a DNA encoding the cloned or determinedprotein of the present invention. Such a polynucleotide (nucleic acid)can be hybridized with an RNA of a protein gene of the present inventionto inhibit synthesis or activities of the RNA, or regulate or controlexpression of a protein gene of the present invention via interactionwith an RNA related to a protein of the present invention. Apolynucleotide complementary to a selected sequence in the RNA relatedto a protein of the present invention and a polynucleotide specificallyhybridizable with the RNA related to a protein of the present inventionare useful for regulating or controlling in vivo or in vitro expressionof a protein gene of the present invention, and for treatment ordiagnosis of a disease. The term “corresponding to” as used herein meanshomology or complementation to a particular sequence of a nucleotideincluding a gene, a nucleotide sequence or a nucleic acid. The term“corresponding to” in terms of relationship between a nucleotide, anucleotide sequence or a nucleic acid and a peptide (protein) generallyrefers to an amino acid in a protein (peptide), a command derived from asequence of a nucleotide (nucleic acid) or its complementary sequence.Examples of a preferable target domain may include a 5′-terminal hairpinloop, a 5′-terminal 6-base pair repeat, a 5′-terminal untranslationdomain, a protein translation initiating codon, a protein coding domain,an ORF translation stop codon, a 3′-terminal non-translation domain, a3′-terminal palindrome domain and a 3′-terminal hairpin loop in aprotein gene of the present invention, but any domain in a protein geneof the present invention may be selected as a target.

Relationship between a given nucleic acid a polynucleotide complementaryto at least apart of a target domain and between a target and ahybridizable polynucleotide can be called “antisense”. Examples of anantisense polynucleotide include a polynucleotide comprising2-deoxy-D-ribose, a polynucleotide comprising D-ribose, other types ofpolynucleotides as an N-glycoside of purine or pyrimidine base and otherpolymers comprising a non-nucleotide structure (for example, acommercially available protein nucleic acid and a synthetic-sequencespecific nucleic acid polymer) or other polymer having a special bondalthough the polymer comprises a nucleotide having a configuration whichcan accept base pairing or base attachment as observed in a DNA or RNA.These may be a double-stranded DNA, a single-stranded DNA, adouble-stranded RNA, a single-stranded RNA or a DNA:RNA hybrid. They mayfurther include unmodified polynucleotides (or unmodifiedoligonucleotide), those having a known modification such as a tag knownin the art, a capping, methylation, replacement of at least one naturalnucleotide with an analogue and an intramolecular nucleotidemodification; those having a non-charged bond (for example,methylphosphonate, phosphotriester, phosphoramidate and carbamate);those having a charged bond or sulfur-containing bond (for example,phosphorothioate and phosphorodithioate); those having a side chaingroup including a protein (a nuclease, a nuclease inhibitor, toxine, anantibody, a signal peptide and poly-L-lysine) or a sugar (for example, amonosaccharide); those comprising an intercurrent compound (for example,acridine and psoralen); those comprising a chelating compound (forexample, a metal, a radioactive metal, boron and an oxidizing metal);those comprising an alkylating agent; and those comprising a modifiedbond (for example, an α-anomer type nucleic acid). The terms“nucleoside”, “nucleotide” and “nucleic acid” as used herein may includenot only those containing purine and pyrimidine bases but also thosefurther containing another modified heterocyclic base. Such a modifiedsubstance may contain methylated purine and pyrimidine, acylated purineand pyrimidine or other heterocycles. A modified nucleoside and amodified nucleotide may be modified in a sugar moiety; for example, oneor more hydroxyls may be replaced with a halogen or aliphatic group, ormay be converted into another functional group such as ether and amine.

An antisense polynucleotide (nucleic acid) of the present invention isan RNA, DNA or modified nucleic acid (RNA, DNA). Examples of a modifiednucleic acid include, but not limited to, a sulfur derivative orthiophosphate derivative of a nucleic acid and those resistant todecomposition by a polynucleosideamide or an oligonuclesideamide. Anantisense nucleic acid of the present invention may be preferablydesigned, for example, such that the antisense nucleic acid is made morestable in a cell, the antisense nucleic acid has a higher permeabilityin a cell, it has affinity to a target sense chain, or if it is toxic,the antisense nucleic acid is made less toxic.

A variety of such modifications are well known in the art, and have beendisclosed in, for example, J. Kawakami et al., Pharm Tech Japan, Vol. 8,pp. 247, 1992; Vol. 8, pp. 395, 1992; S. T. Crooke et al. ed., AntisenseResearch and Applications, CRC Press, 1993.

An antisense nucleic acid of the present invention may comprise aconverted and/or modified sugar, base and/or bond, and thus may beprovided as a special form such as a liposome and a microsphere, may beapplied in gene therapy or may be provided as an adduct. Examples ofsuch an adduct include a polycationic adduct such as polylysine actingas a neutralizer to a charge in a phosphate structure and a hydrophobicadduct such as a lipid enhancing interaction with a cell membrane orincreasing an uptake of a nucleic acid (for example, a phospholipid andcholesterol). Examples of a lipid preferable for addition includecholesterol and its derivatives (for example, cholesteryl chloroformateand cholic acid). It may be attached to a 3′- or 5′-terminal in anucleic acid via a base, sugar or intramolecular nucleoside bond.Another available group may be, for example, a capping groupspecifically located at a 3′- or 5′-terminal in a nucleic acid forpreventing decomposition by a nuclease such as exonuclease and RNase.Examples of such a capping group include, but not limited to, thoseknown as a hydroxyl-protecting group in the art such as glycolsincluding polyethyleneglycol and tetraethyleneglycol.

Inhibition of activity of antisense nucleic acid can be determined usinga transformant of the present invention, an in vivo or in vitro geneexpression system of the present invention, or an in vivo or in vitrotranslation system in a protein of the present invention. The nucleicacid may be applied to a cell by any of various known methods.

A DNA encoding a protein of the present invention may be labeled by aknown method; for example, isotope labeling, fluorescent labeling (forexample, fluorescent labeling with fluorescein), biotinylation andenzyme labeling.

A DNA fully encoding a protein of the present invention may be cloned byamplification by a known PCR using a synthetic DNA primer comprising apartial nucleotide sequence in a protein of the present invention, orselecting a DNA integrated in an appropriate vector by hybridizationwith that labeled with a DNA fragment or synthetic DNA encoding apartial or full-length of a protein of the present invention.Hybridization may be conducted by, for example, a method described inMolecular Cloning 2nd, J. Sambrook et al., Cold Spring Harbor Lab.Press, 1989. When using a commercially available library, hybridizationcan be conducted in accordance with an attached manual.

A sequence of a DNA may be transformed using a known kit such asMutan™-super Express Km (Takara Shuzo Co., Ltd.), Mutan™-K (Takara ShuzoCo., Ltd.), by a known method such as ODA-LA PCR, Gapped duplex methodand Kunkel method or variation thereof.

Depending on an application, a DNA encoding a cloned peptide may be usedas such or if desired used after digestion by a restriction enzyme oraddition of a linker. The DNA may have ATG as a translation initiatingcodon at the 5′-terminal and TAA, TGA or TAG as a translation stop codonat the 3′-terminal. The translation initiating codon or the translationstop codon may be added using an appropriate synthetic DNA adapter.

An expression vector of a protein of the present invention may beproduced by, for example, (i) excising a desired DNA fragment from a DNAencoding a protein of the present invention and (ii) ligating the DNAfragment in the downstream of a promoter in an appropriate expressionvector.

Examples of a vector which can be used include plasmids derived from E.coli (for example, pBR322, pBR325, pUC12 and pUC13); plasmids derivedfrom Bacillus subtilis (for example, pUB110, pTP5 and pC194); plasmidsderived from an yeast (for example, pSH19 and pSH15); bacteriophagessuch as λ-phage; mammalian viruses such as retroviruses, vacciniaviruses and baculoviruses; pA1-11; pXT1; pRc/CMV; pRc/RSV; andpcDNAI/Neo.

A promoter used in the present invention may be any promoter appropriateto a host used in gene expression. For example, when using a mammaliancell as a host, an SR a-promoter, an SV40 promoter, an HIV-LTR promoter,a CMV promoter or an HSV-TK promoter may be used.

Among these, it is preferable to use a CMV (cytomegalovirus) promoter oran SR α-promoter. When a host is an Escherichia coli, a trp promoter, alac promoter, a recA promoter, a λ-PL promoter, an lpp promoter and a T7promoter are preferable; when a host is a Bacillus, an SPO1 promoter, anSPO2 promoter and a penP promoter are preferable; when a host is anyeast, a PHO5 promoter, aPGK promoter, a GAP promoter and an ADHpromoter are preferable. When a host is an insect cell, a polyhetrinpromoter and a P10 promoter are preferable.

If desired, another expression vector can be used, including thosecomprising an enhancer, a splicing signal, a poly-A addition signal, aselection marker and/or an SV40 replication origin (hereinafter,sometimes referred to as “SV40ori”). Examples of a selection markerinclude dihydrofolate reductase (hereinafter, sometimes referred to as“dhfr”) gene [methotrexate (MTX) resistant], an ampicillin resistantgene (hereinafter, sometimes referred to as “Ampr”) and a neomycinresistant gene (hereinafter, sometimes referred to as “Neor”, G418resistant). In particular, when using the dhfr gene as a selectionmarker using a dhfr gene deleted Chinese Hamster cell, integration of adesired gene may be selected using a thymidine deficient medium.

As a selection marker, a reporter gene or drug resistance gene is used(New Biochemical Experimental Lectures (Shin Seikagaku Jikken Koza) 2,nucleic acid III, 3.6 Mammalian cell expression vector, p84-103).

Examples of a combination of a drug resistance gene and a drug which canbe used include:

(1) a combination of a puromycin-N-acetyltransferase gene and puromycin;

(2) a combination of an aminoglycoside phosphotransferase gene (APH) andG418;

(3) a combination of a hygromycin-B phosphotransferase gene (HPH) andhygromycin-B; and

(4) a combination of xanthine-guanine phosphoribosyltransferase (XGPRT)and mycophenolate.

When a parent cell strain is a hypoxanthine-guaninephosphoribosyltransferase (HGPRT) or thymidinekinase (TK) deficientstrain, a combination of these genes and HAT (hypoxanthine, aminopterinand thymidine) may be used.

Dihydrofolate reductase or ampicillin resistance gene may be used as aselection marker gene.

If necessary, a signal sequence appropriate for a host is added to theN-terminal of a protein of the present invention. When a host is anEscherichia coli, a PhoA signal sequence and an OmpA signal sequence maybe used; when a host is a Bacillus, an α-amylase signal sequence and asubtilisin signal sequence may be used; when a host is an yeast, an MFαsignal sequence and an SUC2 signal sequence may be used; when a host isa mammalian cell, an insulin signal sequence, an α-interferon signalsequence and an antibody-molecule signal sequence may be used.

A vector comprising a DNA encoding a protein of the present inventionthus constructed may be used to produce a transformant.

A host may be, for example, an Escherichia coli, a Bacillus, an yeast,an insect cell, an insect or a mammalian cell.

Examples of an Escherichia coli include Escherichia coli K12 DH1 [Proc.Natl. Acad. Sci. USA), Vol. 60, 160 (1968)], JM103 [Nucleic AcidsResearch, Vol. 9, 309 (1981)], JA221 [Journal of Molecular Biology, Vol.120, 517 (1978)], HB101 [Journal of Molecular Biology, Vol. 41, 459(1969)] and C600 [Genetics, Vol. 39, 440 (1954)].

Examples of a Bacillus include Bacillus subtilis MI114 [Gene, Vol. 24,255 (1983)] and 207-21 [Journal of Biochemistry, Vol. 95, 87 (1984)].

Examples of an yeast include Saccharomyces cerevisiae AH22, AH22R⁻,NA87-11A, DKD-5D and 20B-12; Schizosaccharomyces pombe NCYC1913 andNCYC2036; and Pichia pastoris KM71.

In terms of an insect cell, when the virus is AcNPV, an established cellderived from armyworm larva (Spodoptera frugiperda cell; Sf cell), anMG1 cell derived from a midgut of Trichoplusia ni, a High Five™ cellderived from an egg of Trichoplusia ni, a cell derived from Mamestrabrassicae or a cell derived from Estigmena acrea may be used. When thevirus is BmNPV, an established cell derived from silkworm (Bombyx mori Ncell; BmN cell) may be used. Examples of the Sf cell which can be usedinclude an Sf9 cell (ATCC CRL1711) and an Sf21 cell, which have beendescribed in Vaughn, J. L. et al., In Vivo, 13, 213-217 (1977)).

Examples of an insect include silkworm larvae [Maeda et al., Nature,Vol. 315, 592 (1985)].

Examples of a mammalian cell include a simian cell COS-7 (COS7), Vero, aChinese Hamster cell CHO (hereinafter, referred to as “CHO cell”), adhfr-gene deleted Chinese Hamster cell CHO (hereinafter, referred to as“CHO(dhfr⁻) cell”), a murine L cell, a murine AtT-20, a murine myelomacell, a rat GH3 and a human FL cell.

An Escherichia coli may be transformed in accordance with, for example,a method described in Proc. Natl. Acad. Sci. USA), Vol. 69, 2110 (1972)or Gene, Vol. 17, 107 (1982).

A Bacillus may be transformed in accordance with, for example, a methoddescribed in Molecular & General Genetics), Vol. 168, 111 (1979).

An yeast may be transformed in accordance with, for example, a methoddescribed in Methods in Enzymology, Vol. 194, 182-187 (1991) or Proc.Natl. Acad. Sci. USA, Vol. 75, 1929 (1978).

An insect cell or an insect may be transformed in accordance with, forexample, a method described in Bio/Technology, 6, 47-55 (1988).

A mammalian cell may be transformed in accordance with, for example, amethod described in Cell Technology Extra Issue 8, New CellTechnological Experiment Protocols. 263-267 (1995) (Shuju Co. Ltd.) orVirology, Vol. 52, 456 (1973).

Thus, a transformant which has been transformed with an expressionvector comprising a DNA encoding a protein of the present invention canbe obtained.

When culturing a transformant for which a host is an Escherichia coli orBacillus, an appropriate medium used for culturing is a liquid mediumcontaining a carbon source, a nitrogen source, inorganic materials andso on needed for growing the transformant. Examples of a carbon sourceinclude glucose, dextrin, soluble starch and sucrose. Examples of anitrogen source include inorganic and organic materials such as ammoniumsalts, nitrates, corn steep liquor, peptone, casein, meat extract,soybean cake and potato extract. Examples of an inorganic materialinclude calcium chloride, sodium dihydrogenphosphate and magnesiumchloride. An yeast extract, vitamins and/or growth accelerating factorsmay be added. The medium desirably has a pH of about 5 to 8.

A preferable example of a medium for culturing an Escherichia coli maybe an M9 medium containing glucose and casamino acid [Miller, Journal ofExperiments in Molecular Genetics, 431-433, Cold Spring HarborLaboHumanory, New York, 1972]. If necessary, an additional agent such as3β-indolylacrylic acid may be added for efficient promoter activity.

When a host is an Escherichia coli, culturing is generally conducted atabout 15 to 43° C. for about 3 to 24 hours under, if necessary, aerationand/or agitation.

When a host is a Bacillus, culturing is generally conducted at about 30to 40° C. for about 6 to 24 hours under, if necessary, aeration and/oragitation.

When culturing a transformant whose host is an yeast, examples of aculture medium which can be used include a Burkholder minimum medium[Bostian, K. L. et al, Proc. Natl. Acad. Sci. USA, Vol. 77, 4505 (1980)]and an SD medium containing 0.5% of casamino acid [Bitter, G. A. et al.,Proc. Natl. Acad. Sci. USA, Vol. 81, 5330 (1984)]. A pH of the medium ispreferably adjusted to about 5 to 8. Culturing is generally conducted atabout 20 to 35° C. for about 24 to 72 hours under, if necessary,aeration and/or agitation.

When culturing a transformant in which a host is an insect cell orinsect, a culture may be a Grace's Insect Medium (Grace, T. C. C.,Nature, 195, 788 (1962)) containing additives such as 10% decomplementedbovine serum as appropriate. A pH of the medium is preferably adjustedto about 6.2 to 6.4. Culturing is generally conducted at about 27° C.for about 3 to 5 days under, if necessary, aeration and/or agitation.

When culturing a transformant in which a host is a mammalian cell,examples of a culture include an MEM medium containing about 5 to 10%fetal bovine serum [Science, Vol. 122, 501 (1952)], a DMEM medium[Virology, Vol. 8, 396 (1959)], an RPMI 1640 medium [the Journal of theAmerican Medical Association, Vol. 199, 519 (1967)] and a 199 medium[Proceeding of the Society for the Biological Medicine, Vol. 73, 1(1950)]. A pH of the medium is preferably adjusted to about 6 to 8.Culturing is generally conducted at about 30 to 40° C. for about 15 to60 hours under, if necessary, aeration and/or agitation.

As described above, a protein of the present invention may be producedin an intracellular, cell-membrane or extracellular region of atransformant cell.

A protein of the present invention can be isolated and purified from theabove culture by, for example, the following method.

A protein of the present invention can be extracted from culturedbacteria or cells by, as appropriate, a method where after culturing,the bacteria or the cells are collected by a known procedure, they aresuspended in a proper buffer, the bacteria or the cells are lysed usingultrasonic, lysozyme and/or freezing and thawing, then they arecentrifuged or filtrated to give a crude extract of a protein of thepresent invention. The buffer may contain a protein modifier such asurea and guanidine hydrochloride and a surfactant such as Triton X-100™.When a peptide is secreted into a culture medium, the bacteria or thecells are separated from the supernatant by a known method afterculturing, and then the supernatant is collected.

A protein of the present invention contained in the culture supernatantor the extract thus obtained may be purified by an appropriatecombination of known separation/purification methods. Examples of suchknown separation/purification methods include methods utilizing asolubility such as salting out and solvent precipitation; methods mainlyutilizing a molecular weight difference such as dialysis,ultrafiltration, gel filtration and SDS-polyacrylamide gelelectrophoresis; methods utilizing a charge difference such asion-exchange chromatography; methods utilizing specific affinity such asaffinity chromatography; method utilizing a hydrophobicity differencesuch as reverse phase high performance liquid chromatography; andmethods an isoelectric point difference such as isoelectric focusing.

When the protein of the present invention thus obtained is a free form,it can be converted into a salt by a known method or a modificationthereof. In reverse, when it is obtained as a salt, it can be convertedinto a free form or another salt by a known method or a modificationthereof.

Before or after purification, a protein of the present inventionproduced by a recombinant may be attacked by an appropriate proteinmodifying enzyme to be modified as appropriate or to partially remove apeptide. Examples of a protein modifying enzyme include trypsin,chymotrypsin, arginyl-endopeptidase, protein kinase and glycosidase.

An antibody to a protein of the present invention (hereinafter,sometimes simply referred to as “an antibody of the present invention”)may be either polyclonal or monoclonal as long as it is an antibodywhich can recognize an antibody to a protein of the present invention.

The antibody to a protein of the present invention can be produced by aknown method for preparing an antibody or antiserum using a protein ofthe present invention as an antigen.

Preparation of a Monoclonal Antibody

(a) Preparation of a Monoclonal Antibody Producing Cell

A protein of the present invention is applied alone or in combinationwith a carrier and a diluent to a site in a warm-blooded animal where anantibody can be produced. For application, a Freund's complete orincomplete adjuvant may be applied for improving antibody-producingability. It is generally applied once in 2 to 6 weeks and about 2 to 10times in total. Examples of a warm-blooded animal include monkey,rabbit, dog, guinea pig, mouse, rat, sheep, goat and poultry, preferablymouse and rat.

For preparing a monoclonal antibody producing cell, an individualexhibiting an antibody titer from a warm-blooded animal immunized by anantigen such as mouse is selected and a spleen or lymph node is isolated2 to 5 days after final immunization. An antibody producing cellcontained in the isolated may be fused to a myeloma cell of the same ora different animal to prepare a monoclonal antibody producing hybridoma.An antibody titer in an antiserum can be determined by, for example,reacting a labeled peptide described later with the antiserum and thenmeasuring a label activity bound to an antibody. Fusion may be conductedby a known method such as a Kaehler-Milstein method [Nature, 256, 495(1975)]. Examples of a fusion accelerator include polyethyleneglycol(PEG) and Sendai virus, preferably PEG.

Examples of a myeloma cell include those of a warm-blooded animal suchas NS-1, P3U1, SP2/0 and AP-1, preferably P3U1. A preferable numberratio of an antibody producing cell (spleen cell) to a myeloma cell isabout 1:1 to 20:1, PEG (preferably PEG1000 to PEG6000) is added to aconcentration of about 10 to 80%, and the cell fusion can be efficientlyconducted by incubation at 20 to 40° C., preferably 30 to 37° C. for 1to 10 min.

A monoclonal antibody producing hybridoma can be screened by any ofvarious methods; for example, by adding a hybridoma culture supernatantto a solid phase (e.g., microplate) in which a peptide (protein) antigenhas been adsorbed directly or in combination with a carrier, then addingan anti-immunoglobulin antibody (when a cell used for cell fusion is amurine cell, an anti-mouse immunoglobulin antibody is used) or protein Alabeled with a radioactive agent or enzyme, and finally detecting amonoclonal antibody bound to the solid phase, or alternatively by addinga hybridoma culture supernatant to a solid phase in which ananti-immunoglobulin antibody or protein A has been adsorbed, adding apeptide labeled with a radioactive agent or enzyme, and finallydetecting a monoclonal antibody bound to the solid phase.

A monoclonal antibody can be selected by a known method or itsmodification. It can be generally using a medium for a mammalian cellcontaining HAT (hypoxanthine, aminopterin and thymidine). A medium forselection and breeding may be any medium in which a hybridoma can begrown; for example, an RPMI 1640 medium containing 1 to 20%, preferably0 to 20% fetal bovine serum, a GIT medium containing 1 to 10% fetalbovine serum (Wako Pure Chemicals Co. Ltd.) and a serum-free medium forculturing a hybridoma (SFM-101, Nissui Pharmaceutical Co., Ltd.). Aculturing temperature is generally 20 to 40° C., preferably about 37° C.A culturing period is generally 5 days to 3 weeks, preferably 1 to 2weeks. Culturing may be generally conducted under 5% gaseous carbondioxide. An antibody titer in a hybridoma culture supernatant can bedetermined as described for determination of an antibody titer in theabove antiserum.

(b) Purification of a Monoclonal Antibody

A monoclonal antibody can be separated and purified by a known methodincluding separation/purification methods for an immunoglobulin such assalting out, alcohol precipitation, isoelectric precipitation,electrophoresis, an adsorption and desorption method with an ionexchanger (for example, DEAE), ultracentrifugation, gel filtration, anda specific purification in which an antibody is exclusively collected byan activated adsorbent such as an antigen-binding solid phase, protein Aor protein G for dissociating the bond to obtain the antibody.

Preparation of a Polyclonal Antibody

A polyclonal antibody of the present invention can be prepared inaccordance with a known method or its modification. For example, animmunogen (peptide antigen) or its complex with a carrier protein isprepared, a warm-blooded animal is immunized with it as described for apreparation process for the above monoclonal antibody, a productcontaining an antibody to a protein of the present invention iscollected from the immunized animal, and after separation andpurification, the antibody can be prepared.

In terms of a complex of an immunogen and a carrier protein forimmunizing a warm-blooded animal, any type of carrier proteins and anymix ratio of the carrier to a hapten may be employed as long as anantibody can be efficiently produced to a hapten immunized bycrosslinking to the carrier; for example, about 0.1 to 20 parts byweight, preferably about 1 to 5 parts by weight of bovine serum albumin,bovine thyroglobulin, hemocyanin or the like is coupled to 1 part byweight of the hapten.

Various condensing agents may be used for coupling of the hapten withthe carrier, including glutaraldehyde, carbodiimide, activated maleimideester and activated ester reagents having a thiol and/or a dithiopyridylgroups.

A condensation product is applied alone or in combination of a carrierand a diluent, to a site in a warm-blooded animal where an antibody canbe produced. For application, a Freund's complete or incomplete adjuvantmay be applied for improving antibody-producing ability. It is generallyapplied once in 2 to 6 weeks and about 3 to 10 times in total.

A polyclonal antibody can be collected from blood or ascites, preferablyblood of a warm-blooded animal immunized as described above.

A polyclonal antibody titer in an antiserum can be determined asdescribed for determination of an antibody titer in an antiserum. Thepolyclonal antibody can be separated and purified as described forseparation and purification of the immunoglobulin in the course ofseparation/purification of the above monoclonal antibody.

An antisense DNA comprising a nucleotide sequence complementary orsubstantially complementary to a DNA encoding a protein of the presentinvention (hereinafter, the latter DNA is sometimes referred to as “aDNA of the present invention”, and the former antisense DNA is sometimesreferred to as “antisense DNA”) may be any antisense DNA comprising anucleotide sequence complementary or substantially complementary to aDNA of the present invention and capable of inhibiting expression of theDNA.

A nucleotide sequence substantially complementary to a DNA of thepresent invention may be, for example, a nucleotide sequence having ahomology of about 70% or more, preferably about 80% or more, morepreferably 90% or more, most preferably about 95% or more with the fullor partial nucleotide sequence complementary to a DNA of the presentinvention (i.e., a complementary chain to a DNA of the presentinvention). Particularly preferred is an antisense DNA having a homologyof about 70% or more, preferably about 80% or more, more preferably 90%or more, most preferably about 95% or more with a complementary chain toa nucleotide sequence of a domain encoding the N-terminal site of aprotein of the present invention (for example, a nucleotide sequencenear an initiating codon) in the full nucleotide sequence of thecomplementary chain to a DNA of the present invention. Such an antisenseDNA can be prepared using, for example, a known DNA synthesizer.

There will be described applications of an oncogenic protein accordingto the present invention (partial peptide, including a salt), a DNA ofthe present invention, a antibody of the present invention and anantisense DNA of the present invention.

(2) Screening Method for a Compound Promoting or Inhibiting Expressionof an Oncogenic Protein According to the Present Invention

An oncogenic protein of the present invention, an oligonucleotide of thepresent invention, a transformant of the present invention or anantibody of the present invention can be used for a screening method fora compound promoting or inhibiting expression of a protein of thepresent invention.

Specifically, the present invention provides

(i) a method for screening a compound promoting or inhibiting expressionof an oncogenic protein of the present invention comprising determiningand comparing the amount of expression of an oncogenic protein of thepresent invention or the amount of an mRNA encoding an oncogenic proteinof the present invention when culturing a cell or tissue which canexpress an oncogenic protein of the present invention in the presence orabsence of a test compound.

Examples of a cell or tissue which can express an oncogenic protein ofthe present invention include a human-derived cell, a warm-bloodedanimal (e.g., guinea pig, rat, mouse, poultry, rabbit, pig, sheep,bovine and monkey) cell (for example, neural cell, endocrine cell,neuroendocrine cell, glia cell, pancreatic β-cell, marrow cell, hepaticcell, splenic cell, mesangium cell, epidermal cell, epithelial cell,endothelial cell, fibroblast, fibrocyte, myocyte, adipocyte, immunocyte(e.g., macrophage, T-cell, B-cell, natural killer cell, mast cell,neutrophile, basophilic cell, acidophilic leucocyte, monocyte, dendriticcell), mega karyocyte, synoviocyte, cartilage cell, osteocyte,osteoblast, osteoclast, mammary glandular cell, intersitial cell andtheir precursor cells, stem cells and carcinoma cells, as well as alltissues where any of these cells is present such as brain, brain sites(for example, olfactory bulb, amygdaloid nucleus, basal cistern,hippocampus, optic thalamus, hypothalamus, cerebral cortex, medulaoblongata, cerebellum), spinal cord, pituitary gland, stomach, pancreas,kidney, liver, genital gland, hyroid gland, gallbladder, bone marrow,adrenal gland, skin, muscle, lung, gastrointestinal tract (for example,large intestine and small intestine), blood vessel, heart, thymus gland,spleen, salivary gland, peripheral blood, prostate gland, testicle(spermary), ovary, placenta, uterus, bone, cartilage, joint and skeletalmuscle. Here, an established cell or primary culture system may be used.In particular, it is desirable to use the above transformant cell of thepresent invention.

A method for culturing a cell which can express a protein of the presentinvention is as described for culturing the above transformant of thepresent invention.

A test compound may be, in addition to the above test compounds, a DNAlibrary.

The amount of expression of a cancer cell of the present invention canbe determined a known method such as an immunochemical method using, forexample, an antibody, or alternatively an mRNA encoding an oncogenicprotein of the present invention can be determined by a known methodusing Northern hybridization, RT-PCR or TaqMan PCR.

The amounts of expression of an mRNA can be compared by hybridization inaccordance with a known method or its modification, for example, amethod described in Molecular Cloning 2nd, J. Sambrook et al., ColdSpring Harbor Lab. Press, 1989.

Specifically, the amount of an mRNA encoding an oncogenic protein of thepresent invention is determined according to a known method, i.e., bycontacting an RNA extracted from a cell with a polynucleotide of thepresent invention or its part or an antisense polynucleotide of thepresent invention and then measuring the amount of mRNA bound to apolynucleotide of the present invention or its part or an antisensepolynucleotide of the present invention. A polynucleotide of the presentinvention or its part or an antisense polynucleotide of the presentinvention can be labeled with, for example, a radioisotope or dye tofacilitate determination of the amount of mRNA bound to a polynucleotideof the present invention or its part or an antisense polynucleotide ofthe present invention. Examples of a radioisotope include ³²P and ³H,and examples of a dye include fluorochromes such as fluorescein, FAM (PEBiosystems Inc.), JOE (PE Biosystems Inc.), TAMRA (PE Biosystems Inc.),ROX (PE Biosystems Inc.), Cy5 (Amersham Inc.) and Cy3 (Amersham Inc.).

The amount of an mRNA can be determined by transforming an RNA extractedfrom a cell into cDNA using a reverse transcriptase and then measuringthe amount of amplified cDNA by PCR using a polynucleotide of thepresent invention or its part or an antisense polynucleotide of thepresent invention as a primer.

Thus, a test compound which increases the amount of an mRNA encoding anoncogenic protein of the present invention can be selected as a compoundpromoting expression of an oncogenic protein of the present invention,while a test compound which reduce the amount of an mRNA encoding anoncogenic protein of the present invention can be selected a compoundinhibiting expression of an oncogenic protein of the present invention.

The present invention also provides

(ii) a method for screening a compound promoting or inhibiting promoteractivity comprising determining and comparing a reporter activity whenculturing, in the presence or absence of a test compound, a transformantobtained by transforming with a recombinant DNA in which a reporter geneis ligated to the downstream of a promoter or enhancer domain for a geneencoding an oncogenic protein of the present invention.

Examples of a reporter gene which can be used include lacZ(β-galactosidase gene), chloramphenicol acetyltransferase (CAT),luciferase, growth factors, β-glucuronidase, alkaline phosphatase, Greenfluorescent protein (GFP) and β-lactamase.

By determining the amount of a reporter gene product (e.g., mRNA andprotein) using a known method, a test compound which increases theamount of the reporter gene product can be selected as a compoundcontrolling (particularly, promoting) promoter or enhancer activity of aprotein of the present invention, i.e., as a compound promotingexpression of a protein of the present invention. On the contrary, atest compound which reduces the amount of the reporter gene product canbe selected as a compound controlling (particularly, inhibiting)promoter or enhancer activity of a protein of the present invention,i.e., as a compound inhibiting expression of a protein of the presentinvention.

A test compound may be as described above.

A vector comprising a reporter gene can be constructed or assayed by aknown technique (For example, see Molecular Biotechnology 13, 29-43,1999).

Since a compound inhibiting expression of an oncogenic protein of thepresent invention can inhibit biological activities of an oncogenicprotein of the present invention, it is useful as a safe and low-toxicmedical drug for inhibiting biological activities of an oncogenicprotein of the present invention. Specifically, it is useful as aprophylactic or therapeutic drug for a cancer such as pulmonary cancer,renal cancer, hepatic carcinoma, non-small cell pulmonary cancer,ovarian cancer, prostatic cancer, gastric cancer, bladder cancer, breastcancer, cervical cancer, colonical cancer, rectal cancer and pancreaticcancer, particularly cervical cancer.

A compound or its salt obtained using a screening method or screeningkit of the present invention may be, for example, a compound selectedfrom the group consisting of a peptide, a protein, a non-peptidecompound, a synthetic compound, a fermentation product, a cell extract,a plant extract, an animal-tissue extract and plasma. A salt of thecompound may be as described for a salt of a peptide derived from anoncogenic protein of the present invention.

When using a compound obtained using a screening method or screening kitaccording to the present invention as the above therapeutic orprophylactic agent, it can be used as usual. For example, as describedfor a medicine comprising a protein of the present invention, it can beprepared as a tablet, a capsule, an elixir, a microcapsule, a sterilesolution and a suspension.

A dose of the compound or its salt depends on various factors such asits effect, a target disease, a recipient and a delivery route. Forexample, in oral administration of a compound inhibiting expression of aprotein of the present invention for treating a cancer, its dose isabout 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferablyabout 1.0 to 20 mg per day to a normal adult (weight: 60 kg). Inparenteral administration, although a dose of the compound depends onvarious factors such as a recipient and a target disease, its dose issuitably about 0.01 to 30 mg, preferably about 0.1 to 20 mg, morepreferably about 0.1 to 10 mg per day by intravenous injection when acompound inhibiting expression of a protein of the present invention isadministered to a normal adult (weight: 60 kg) for treating a cancer.For another animal, an amount converted to a weight of 60 kg may beadministered.

(3) Assay of an Oncogenic Protein of the Present Invention

An antibody of the present invention can specifically recognize anoncogenic protein of the present invention, so that it can be used forassaying the oncogenic protein of the present invention in a testsolution, particularly an assay by sandwich immunity measuring method.

Thus, the present invention provides

(i) a process for assaying an oncogenic protein of the present inventionin a test solution comprising the steps of competitively reacting anantibody of the present invention with the test solution and a labeledprotein of the present invention and then determining a proportion ofthe labeled protein of the present invention bound to the antibody; and

(ii) a process for assaying an oncogenic protein of the presentinvention in a test solution comprising the steps of simultaneously orsequentially reacting the test solution with an antibody of the presentinvention insolubilized on a carrier and another labeled antibody of thepresent invention and then determining an activity of the labeling agenton the insolubilized carrier.

In the assay described in (ii), it is desirable that one antibody is anantibody recognizing the N-terminus in an oncogenic protein of thepresent invention, while the other antibody is an antibody reacting withthe C-terminus in an oncogenic protein of the present invention.

A monoclonal antibody to an oncogenic protein of the present inventioncan be used for assaying an oncogenic protein of the present inventionor detection using, for example, tissue staining. For these purposes, anantibody molecule itself can be used, or alternatively, an F(ab′)₂, Fab′or Fab fraction of the antibody molecule can be used.

There are no particular restrictions to a procedure for assaying anoncogenic protein of the present invention using an antibody of thepresent invention, and it may be any procedure whereby the amount of anantibody, antigen or antibody-antigen complex corresponding to theamount of an antigen (for example, the amount of a peptide) in ameasured solution can be detected by chemical or physical means and adesired value can be calculated using a standard curve plotted using thestandard solution containing a known amount of the antigen. For example,nephelometry, a competition assay, an immunoradiometric assay and asandwich assay are suitably used, and in terms of sensitivity andspecificity, a sandwich assay described below is particularlypreferable.

Examples of a labeling agent used in an assay using such a labelingmaterial include a radioisotope, an enzyme, a fluorescent substance anda luminescent material. Examples of a radioisotope include [¹²⁵I],[¹³¹I], [³H] and [¹⁴C]. Among these, a stable enzyme exhibiting a largespecific activity is preferable, including β-galactosidase,β-glucuronidase, alkaline phosphatase, peroxidase and malatedehydrogenase. Examples of a fluorescent substance include fluorescamineand fluorescein isothiocyanate. Examples of a luminescent materialinclude luminol, luminol derivatives, luciferin and lucigenin. Abiotin-avidin system may be used for binding an antibody or antigen witha labeling agent.

An antigen or antibody can be insolubilized using physical adsorption,or using a chemical bond commonly used for insolubilizing orimmobilizing a peptide or enzyme. Examples of a carrier includeinsoluble polysaccharides such as agarose, dextran and cellulose;synthetic resins such as polystyrene, polyacrylamide and silicones; andglasses.

In a sandwich assay, an insolubilized monoclonal antibody of the presentinvention is reacted with a test solution (first reaction), and thenreacted with another labeled monoclonal antibody of the presentinvention (second reaction). Then, an activity of the labeling agent onthe insolubilized carrier can be determined to assay the amount of aprotein of the present invention in the test solution. The first and thesecond reactions may be conducted in the reverse order, simultaneouslyor sequentially. A labeling agent and an insolubilizing method may be asdescribed above. In an immunity measuring method using a sandwich assay,it is not necessary to use only one antibody for a solid phase or forlabeling, but a mixture of two or more antibodies may be used for, forinstance, improving measurement sensitivity.

In determination of an oncogenic protein of the present invention by asandwich assay according to the present invention, monoclonal antibodiesof the present invention used in the first and the second reactions ispreferably antibodies having a different site to which the oncogenicprotein of the present invention is bound. Specifically, in terms ofantibodies used in the first and the second reactions, when the antibodyused in the second reaction recognizes the C-terminus in the oncogenicprotein of the present invention, the antibody used in the firstreaction is an antibody which recognizes a site other than theC-terminus, for example the N-terminus.

A monoclonal antibody of the present invention may be used a measurementsystem other than a sandwich assay, such as a competitive assay,immunometry and nephelometry.

In a competitive assay, an antigen in a test solution and a labeledantigen are competitively reacted with an antibody, the unreactedlabeled antigen (F) is separated from the bound labeled antigen (B) (B/Fseparation) and the labeled amount of either B or F is determined toassay the antigen amount in the test solution. This reaction assay canbe conducted by a liquid phase method where a soluble antibody is usedas an antibody and polyethylene glycol and a secondary antibody to theabove antibody are used in B/F separation, or alternatively by a solidphase method where a immobilized antibody is used as the first antibodyor the first antibody is a soluble one and the second antibody is aimmobilized antibody.

In immunometry, an antigen in a test solution and a immobilized antigenare competitively reacted with a given amount of a labeled antibody, thesolid and the liquid phases are separated, or alternatively an antigenin a test solution is reacted with an excessive amount of a labeledantibody, a immobilized antigen is added to allow the unreacted labeledantibody to bind to the solid phase and then the solid and the liquidphases are separated. Then, the label amount in either phase isdetermined to assay the antigen amount in the test solution.

Nephelometry determines the amount of an insoluble precipitate resultingfrom an antigen-antibody reaction in a gel or solution. Even when theamount of an antigen in a test solution is small so that a small amountof precipitate is formed, laser nephelometry utilizing laser scatteringis suitably used.

For applying the individual immunological measuring methods to an assayof the present invention, special conditions or operations are notnecessary. A measuring system for a protein of the present invention maybe constructed in the light of common conditions and operations in theindividual methods with modifications known to the skilled in the art.Details in these common technical procedures will be found in variousreviews and textbooks.

Such literatures may be, for example, “Radioimmunoassay”, ed. by HiroshiIrie, Kodansha, published in 1974; “Radioimmunoassay, 2^(nd),”, ed. byHiroshi Irie, Kodansha, published in 1979; “Enzyme Immunoassay”, ed. byEiji Ishikawa et al., Igaku-Shoin, published in 1978; “EnzymeImmunoassay, 2^(nd) Edition”, ed. by Eiji Ishikawa et al., Igaku-Shoin,published in 1982; “Enzyme Immunoassay, 3^(rd) Edition”, ed. by EijiIshikawa et al., Igaku-Shoin, published in 1987; “Methods inENZYMOLOGY”, Vol. 70 (Immunochemical Techniques (Part A)); ibid., Vol.73 (Immunochemical Techniques (Part B)); ibid., Vol. 74 (ImmunochemicalTechniques (Part C)); ibid., Vol. 84 (Immunochemical Techniques (Part D:Selected Immunoassays)); ibid., Vol. 92 (Immunochemical Techniques (PartE: Monoclonal Antibodies and General Immunoassay Methods)); and ibid.,Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology andMonoclonal Antibodies)) (these have been published by Academic Press).

When reduction in a level of an oncogenic protein of the presentinvention is detected by assaying the level using an antibody of thepresent invention, for example, it leads to a diagnosis that the subjecthas a disease associated with insufficiency in the oncogenic protein ofthe present invention or will probably contract the disease in future.

When increase in a level of an oncogenic protein of the presentinvention is detected, for example, it leads to a diagnosis that thesubject has a disease induced by over-expression of the oncogenicprotein of the present invention, including cancers such as pulmonarycancer, renal cancer, hepatic carcinoma, non-small cell pulmonarycancer, ovarian cancer, prostatic cancer, gastric cancer, bladdercancer, breast cancer, cervical cancer, colonical cancer, rectal cancerand pancreatic cancer (in particular, cervical cancer), or will probablycontract the disease in future.

An antibody of the present invention may be used to detect an oncogenicprotein of the present invention present in a sample such as a bodyfluid and a tissue. It can be also used for preparing an antibody columnused for purification of an oncogenic protein of the present invention,detection of a protein of the present invention in each fraction duringpurification and analysis of behavior of a protein of the presentinvention in a test cell.

(4) Gene Diagnostic Agent

A polynucleotide or antisense polynucleotide of the present inventioncan be also, for example, used as a nucleic acid probe for detecting anabnormality (gene defect) in a DNA or mRNA encoding an oncogenic proteinof the present invention mainly in a human. For example, it is useful asa gene diagnostic agent for injury, mutation or reduced expression inthe DNA or the mRNA or over-expression of the mRNA.

The above gene diagnosis using a polynucleotide or antisensepolynucleotide of the present invention can be conducted in accordancewith, for example, known Northern hybridization or PCR-SSCP (See,Genomics, Vol. 5, pp. 874-879 (1989); Proceedings of the NationalAcademy of Sciences of the United States of America, Vol. 86, pp.2766-2770 (1989)).

For example, when reduction in expression of an mRNA is detected byNorthern hybridization, it leads to a diagnosis that the subject has adisease associated with insufficiency of an oncogenic protein of thepresent invention or will probably contract the disease in future.

When over-expression of an mRNA is detected by Northern hybridization,for example, it leads to a diagnosis that the subject probably has adisease induced by over-expression of the oncogenic protein of thepresent invention, including cancers such as pulmonary cancer, renalcancer, hepatic carcinoma, non-small cellpulmonary cancer, ovariancancer, prostatic cancer, gastric cancer, bladder cancer, breast cancer,cervical cancer, colonical cancer, rectal cancer and pancreatic cancer(in particular, cervical cancer), or will probably contract the diseasein future.

(5) Drug Containing an Antisense Polynucleotide

An antisense polynucleotide of the present invention whichcomplementarily binds to a polynucleotide of the present invention (forexample, DNA) and can inhibit expression of the polynucleotide (forexample, DNA) is less toxic and can inhibit in vivo activity of theprotein of the present invention or the polynucleotide of the presentinvention (for example, DNA). Thus, it is useful as a prophylactic ortherapeutic agent for a disease induced by over-expression of theprotein of the present invention, for example, a cancer such aspulmonary cancer, renal cancer, hepatic carcinoma, non-smallcellpulmonary cancer, ovarian cancer, prostatic cancer, gastric cancer,bladder cancer, breast cancer, cervical cancer, colonical cancer, rectalcancer and pancreatic cancer.

When using the antisense polynucleotide as the above prophylactic ortherapeutic agent, the antisense polynucleotide can be formulated asdescribed for the above polynucleotide of the present invention.

The formulation thus obtained is less toxic and can be orally orparenterally administered to a human or mammal (for example, rat,rabbit, sheep, pig, bovine, cat, dog and monkey).

The antisense polynucleotide can be administered as such or incombination with a physiologically acceptable carrier such as anadjuvant for promoting intake, using a gene gun or a catheter such as ahydrogel catheter.

A dose of the antisense polynucleotide varies depending on many factorssuch as a target disease, a recipient and a delivery route, and is, forexample, about 0.1 to 100 mg per day to an adult (weight: 60 kg) when anantisense nucleotide of the present invention is locally administered toan organ (for example, liver, lung, heart and kidney) for treating acancer.

Furthermore, the antisense polynucleotide can be used as a diagnosticnucleotide probe for determining the presence of an oncogene DNA of thepresent invention in a tissue or cell or its expression status.

The present invention also provides

(i) a double strand RNA comprising a part of an RNA encoding a proteinof the present invention or an RNA complementary thereto;

(ii) a drug comprising the double strand RNA;

when appropriate,

(iii) a ribozyme comprising a part of an RNA encoding a protein of thepresent invention; and

(iv) a drug comprising the ribozyme.

These double strand RNA (RNAi; RNA interference method) and the ribozymecan inhibit expression of a polynucleotide (for example, DNA) of thepresent invention as in the above antisense polynucleotide and caninhibit in vivo activities of an oncogenic protein or polynucleotide(for example, DNA) of the present invention. Thus, it is useful as aprophylactic or therapeutic agent for a disease induced byover-expression of the oncogenic protein of the present invention, forexample, a cancer such as pulmonary cancer, renal cancer, hepaticcarcinoma, non-small cellpulmonary cancer, ovarian cancer, prostaticcancer, gastric cancer, bladder cancer, breast cancer, cervical cancer,colonical cancer, rectal cancer and pancreatic cancer, particularlycervical cancer.

The double strand RNA can be designed and produced on the basis of thesequence of the polynucleotide of the present invention in accordancewith a known method (See, for example, Nature, Vol. 411, p. 494, 2001).

The ribozyme can be designed and produced on the basis of thepolynucleotide of the present invention in accordance with a knownmethod (See, for example, TRENDS in Molecular Medicine, Vol. 7, p. 221,2001). For example, it can be produced by ligating a known ribozyme to apart of an RNA encoding a protein of the present invention. An exampleof the part of an RNA encoding a protein of the present invention is apart (RNA fragment) near a restriction site on the RNA of the presentinvention which can be digested by a known ribozyme.

When using the double strand RNA or the ribozyme as the aboveprophylactic or therapeutic agent, it can be formulated and administeredas described for the antisense polynucleotide.

(6) Medical Drug Comprising an Antibody of the Present Invention

An antibody of the present invention capable of neutralizingcancerization activity of an oncogenic protein of the present inventioncan be used as a prophylactic or therapeutic drug for a disease inducedby over-expression of the oncogenic protein of the present invention,for example, a cancer such as pulmonary cancer, renal cancer, hepaticcarcinoma, non-small cell pulmonary cancer, ovarian cancer, prostaticcancer, gastric cancer, bladder cancer, breast cancer, cervical cancer,colonical cancer, rectal cancer and pancreatic cancer, particularlycervical cancer.

The above prophylactic or therapeutic drug for the disease comprisingthe antibody of the present invention can be orally or parenterallyadministered to a human or mammal (for example, rat, rabbit, sheep, pig,bovine, cat, dog and monkey) as such, i.e., as a solution or as anappropriate dosage form of pharmaceutical composition. A dose may varydepending on various factors such as a recipient, a target disease, asymptom and a delivery route, and may be, for example, generally 0.01 to20 mg/kg, preferably 0.1 to 10 mg/kg, more preferably 0.1 to 5 mg/kg ofthe antibody of the present invention for a dose, about once to fivetimes a day, preferably one to three times a day. It can be suitablyadministered by intravenous injection. In another type of parenteral ororal administration, a dose similar to that described above may beemployed. If a symptom is particularly severe, a dose may be furtherincreased, depending on the symptom.

An antibody of the present invention can be administered as such or asan appropriate pharmaceutical composition. A pharmaceutical compositionused for the above administration comprises the above antibody or itssalt and a pharmacologically acceptable carrier, diluent or excipient.Such a composition is provided as a dosage form appropriate for oral orparenteral administration.

Specifically, examples of a composition for oral administration includesolid and liquid dosage forms including tablet such as sugar-coatedtablet and film-coated tablet), pill, granule, powder, capsule such assoft capsule, syrup, emulsion and suspension. Such a composition isprepared by a known process and comprises a carrier, diluent orexcipient commonly used in the art of pharmacy. Examples of a carrier orexcipient for tablet include lactose, starch, sucrose and magnesiumstearate.

Examples of a composition for parenteral administration include aninjection and a suppository. Examples of an injection include anintravenous injection, a subcutaneous injection, an intracutaneousinjection, an intramuscular injection and an intravenous drip injection.Such an injection may be prepared by a known method; for example, bydissolving, suspending or emulsifying the above antibody or its salt ina sterile aqueous or oily liquid commonly used for an injection.Examples of an aqueous liquid for injection include saline and anisotonic solution containing glucose or other adjuvants, which may becombined with an appropriate solubilizing agent including alcohols suchas ethanol; polyols such as propyleneglycol and polyethyleneglycol;nonionic surfactants such as polysorbate 80 and HCO-50 (polyoxyethylene(50 mol) adduct of hydrogenated castor oil). Examples of an oily liquidinclude sesame oil and soybean oil, which may be combined with asolubilizing agent such as benzyl benzoate and benzyl alcohol. Aninjection thus prepared is generally filled in an appropriate ampule. Asuppository used for rectal administration is prepared by mixing theabove antibody or its salt with a common suppository base.

The oral or parenteral pharmaceutical composition is convenientlyprepared in a dosage form suitable for a dose of an active ingredient.Examples of such a unit dosage form include tablet, pill, capsule,injection (ampule) and suppository. Each unit dosage form preferablycontains generally 5 to 500 mg of the above antibody. In particular, aninjection contains 5 to 100 mg, while another dosage form contains 10 to250 mg.

Each of the above compositions may contain other active ingredients aslong as they result in undesirable interaction when being compoundedwith the antibody.

(7) DNA Transferred Animal

The present invention provides a non-human mammal, for example, a“knock-in” animal, having a DNA encoding a human-derived oncogenicprotein of the present invention (hereinafter, referred to as “anoncogene DNA of the present invention”) or its variant DNA.

Specifically, the present invention provides

(1) a non-human mammal having a human-derived oncogene DNA of thepresent invention or its variant DNA (“knock-in” animal);

(2) the “knock-in” animal in which the non-human mammal is a rodent;

(3) the “knock-in” mouse or rat in which the rodent is mouse or rat; and

(4) a recombinant vector comprising the human-derived oncogene DNA ofthe present invention or its variant DNA which can be expressed in thenon-human mammal.

A non-human mammal having a human-derived oncogene DNA or its variantDNA of the present invention (hereinafter, referred to as “a DNAtransferred animal of the present invention”) can be produced bytransferring a desired DNA to an unfertilized egg, a fertilized egg, asperm or a germinal cell including their initial cells, preferably in anembryogenesis stage in development of the non-human mammal (furtherpreferably, in a stage of a single cell or fertilized egg cell andgenerally up to the 8-cell phase) by an appropriate method such as acalcium phosphate method, an electrical pulse method, lipofection,aggregation, microinjection, a particle gun method and a DEAE-dextran.By the DNA transfer method, a desired foreign DNA of the presentinvention may be transferred to a somatic cell, living organ or tissuecell for using it in cell culture or tissue culture. Furthermore, thecell can be fused with the above germinal cell by a known cell-fusionprocess to produce a DNA transferred animal of the present invention.

Examples of a non-human mammal include bovine, pig, sheep, goat, rabbit,dog, cat, guinea pig, hamster, mouse and rat. Among these, preferred isa rodent, particularly mouse (for example, a pure line such as a C57BL/6strain and a DBA2 strain, and a hybrid line such as a B6C3F₁ strain, aBDF₁ strain, a B6D2F₁ strain, a BALB/c strain ad an ICR strain) or rat(for example, Wistar and SD) because its ontogenesis and biologicalcycles are relatively shorter in the light of producing a disease animalmodel and it can be easily bred.

A “mammal” in terms of a recombinant vector which can be expressed in amammal may be, in addition to the above non-human mammal, a human.

A variant DNA to an oncogene derived from a human of the presentinvention refers not to a DNA of a homologue to an oncogene of thepresent invention inherent in a non-human mammal, but an artificiallymutated one.

A variant DNA of the present invention may be a human-derived oncogeneDNA of the present invention whose original nucleotide sequence has beenvaried (for example, mutation) such as a DNA having addition of a base,deletion or substitution with another base, or an abnormal DNA.

An abnormal DNA refers to a DNA expressing a protein which is similar toa normal oncogenic protein of the present invention but has a differentactivity; for example, a DNA expressing a peptide in which an activityof a normal oncogenic protein of the present invention is inhibited.

For transferring an oncogene DNA of the present invention to a targetnon-human animal, it is generally advantageous to use the DNA as a DNAconstruct bound in the downstream of a promoter capable of expressingthe DNA in the non-human animal cell. For example, when transferring ahuman-derived DNA of the present invention, a DNA construct (forexample, a vector) to which a human-derived oncogene DNA of the presentinvention has been bound can be microinjected to a fertilized egg of atarget non-human mammal, for example, a murine fertilized egg, in thedownstream of any of various promoters capable of expressing a DNAderived from any of various non-human mammals exhibiting higher homologyto the DNA of the present invention (for example, rabbit, dog, cat,guinea pig, hamster, rat and mouse), to produce a DNA transferrednon-human mammal capable of highly expressing the oncogene DNA of thepresent invention.

Examples of an expression vector for an oncogenic protein of the presentinvention include a E. coli derived plasmid, a Bacillus subtilis derivedplasmid, an yeast derived plasmid, a bacteriophage such as λ-phage, aretrovirus such as Moloney leukemia virus and a mammalian virus such asa vaccinia virus and a baculovirus, preferably a E. coli derivedplasmid, a Bacillus subtilis derived plasmid and an yeast derivedplasmid.

Examples of a promoter for regulating the DNA expression include

(i) promoters for a DNA derived from a virus (for example, simian virus,cytomegalovirus, Moloney leukemia virus, JC virus, mammary tumor virusand poliovirus);

(ii) promoters derived from various mammals (for example, human, rabbit,dog, cat, guinea pig, hamster, rat and mouse); for example, a promoterfor albumin, insulin II, uroplakin II, elastase, erythropoietin,endothelin, muscle cretine kinase, glial fibrillary acidic protein,glutathione S-transferase, platelet-derived growth factor β, keratin K1,K10 and K14, type I and II collagens, cyclic-AMP dependent proteinkinase βI subunit, dystrophin, tartrate-resistant alkaline phosphatase,atrial sodium diuretic factor, endothelial receptor tyrosine kinase(generally, abbreviated as “Tie2”), sodium potassium adenosinetriphosphatase (Na, K-ATPase), neurofilament light chain,metallothionine I and IIA, metalloproteinase-1 tissue inhibitor, MHCClass I antigen (H-2L), H-ras, renin, dopamin β-hydroxylase, thyroidperoxidase (TPO), peptide chain elongation factor 1α (EF-1α), β-actin,α- and β-myosin heavy chain, myosin light chain 1 and 2, myelin baseprotein, thyroglobulin, Thy-1, immunoglobulin, variable H-chain part(VNP), serum amyloid P component, myoglobin, troponin C, smooth muscleα-actin, preproenkephalin A and vasopressin. Among these, preferred area cytomegalovirus promoter, a promoter of human peptide chain elongationfactor 1α (EF-1α), a human and a poultry β-actin promoters capable ofhigher expression in an entire body.

The above vector preferably comprises a sequence which terminatetranscription of a desired messenger RNA in a DNA transferred non-humanmammal (generally, called “terminator”). For example, DNA sequencesderived from bovine or various mammals can be used, preferably a simianvirus SV40 terminator.

Furthermore, if desired, it may be possible to ligate a splicing signalfor each DNA, an enhancer region or a part of a eukaryotic DNA intron inthe 5′-upstream of the promoter region, between the promoter region anda translation region, or in the 3′-downstream of the translation regionfor higher expression of a desired oncogene DNA.

A translation region of a normal oncogenic protein of the presentinvention can be obtained using, as a starting material, a complementaryDNA prepared as a whole or partial genome DNA from human-derived liver,kidney or thyroid gland cell, a fibroblast-derived DNA and variouscommercially available genome DNA libraries, or by a known process froman RNA derived from liver cell, kidney cell, thyroid gland cell orfibroblast. For producing an abnormal DNA induced by mutation, atranslation region in the normal peptide obtained from the above cell ortissue can be mutated by a point mutation induction method to produce amutant translation region.

The translation region can be produced as a DNA construct which can beexpressed in a DNA transferred animal, by a common DNA engineeringprocedure in which it is ligated to the downstream of the promoter or ifdesired the upstream of a transcription termination site.

Transfer of a human-derived oncogene DNA of the present invention in afertilized egg cell stage is conducted such that the DNA is carried inall of germinal and somatic cells in a target non-human mammal. Presenceof the oncogene DNA of the present invention in germinal cells in aproduced animal after DNA transfer means that all progenies of theproduced animal will carry the oncogene DNA of the present invention inall of their germinal and somatic cells. Offsprings of this type ofanimal inheriting the oncogene DNA of the present invention carry theoncogene DNA of the present invention in all of their germinal andsomatic cells.

A non-human mammal to which a human-derived oncogene DNA of the presentinvention has been transferred can be successively bred under commonbreeding circumstances as a mammal carrying the oncogene DNA, afterconfirming that the mammal stably carries the human-derived oncogene DNAafter mating.

Transfer of a human-derived oncogene DNA of the present invention in afertilized egg cell stage is conducted such that the DNA is carried inall of germinal and somatic cells in a target non-human mammal. Presenceof the human-derived oncogene DNA of the present invention in germinalcells in a produced mammal after DNA transfer means that all offspringsof the produced mammal will successively carry the human-derivedoncogene DNA of the present invention in all of their germinal andsomatic cells. Offsprings of this type of animal inheriting thehuman-derived oncogene DNA of the present invention successively carrythe human-derived oncogene DNA of the present invention in all of theirgerminal and somatic cells.

A homozygote animal having a transduced DNA in both of homologouschromosomes can be produced and the male and the female animals can bemated for successive breeding such that all offsprings successivelycarry the DNA.

In a non-human mammal having a human-derived oncogene DNA of the presentinvention, the normal DNA of the present invention is highly expressed,and an activity of the intrinsic normal DNA may be promoted, sometimesresulting in onset of hyperactivity of the protein of the presentinvention. Thus, it can be used as a model animal for the disease. Forexample, a normal DNA transferred animal of the present invention can beused to elucidate a mechanism of hyperactivity of the oncogenic proteinof the present invention or of a disease associated with the oncogenicprotein of the present invention and investigate a therapy for thesediseases.

Furthermore, since a non-human mammal to which a human-derived oncogeneDNA of the present invention has been transferred shows increase in thefree protein of the present invention, it can be used for screening atherapeutic agent to a disease associated with the oncogenic protein ofthe present invention.

On the other hand, a non-human mammal having an abnormal DNA of thepresent invention can be successively bred under common breedingcircumstances as an animal carrying the DNA, after confirming that theanimal stably carries the transduced DNA after mating. Furthermore, adesired abnormal DNA can be incorporated into the above plasmid to beused as a starting material. A DNA construct with a promoter can beproduced by a common DNA engineering procedure. Transfer of an abnormalDNA of the present invention in a fertilized egg cell stage is conductedsuch that the DNA is carried in all of germinal and somatic cells in atarget mammal. Presence of the abnormal DNA of the present invention ingerminal cells in a produced animal after DNA transfer means that alloffsprings of the produced animal will carry the abnormal DNA of thepresent invention in all of their germinal and somatic cells. Offspringsof this type of animal inheriting the abnormal DNA of the presentinvention carry the abnormal DNA of the present invention in all oftheir germinal and somatic cells. A homozygote animal having atransduced DNA in both of homologous chromosomes can be produced and themale and the female animals can be mated for successive breeding suchthat all offsprings successively carry the DNA.

In a non-human mammal having an abnormal DNA of the present invention,the abnormal DNA of the present invention is highly expressed, and anactivity of the intrinsic normal DNA may be inhibited, sometimesresulting in a deactivation type refractoriness of the protein of thepresent invention. Thus, it can be used as a model animal for thedisease. For example, an abnormal DNA transferred animal of the presentinvention can be used to elucidate a mechanism of the deactivation typerefractoriness of the oncogenic protein of the present invention andinvestigate a therapy for the disease.

As a specific application, an animal highly expressing the abnormal DNAof the present invention can be used as a model forelucidatinginhibition of a normal peptide activity (dominant negative activity) bythe abnormal peptide of the present invention in the deactivation typerefractoriness of the protein of the present invention.

Furthermore, since a non-human mammal to which an abnormal DNA of thepresent invention has been transferred shows increase in the freeoncogenic protein of the present invention, it can be used for screeninga therapeutic agent to the oncogenic protein or its deactivation typerefractoriness.

A DNA transferred animal of the present invention can be used forinvestigating a clinical symptom of a disease associated with anoncogenic protein of the present invention such as a deactivation typerefractoriness of the protein of the present invention. Furthermore, theanimal can give more specific pathologic observation in each organ in adisease model associated with the oncogenic protein of the presentinvention and can contribute to development of a new therapy andinvestigation and treatment of a secondary disease due to the abovedisease.

Each organ can be extracted from a DNA transferred animal of the presentinvention, chopped and treated with a protease such as trypsin to obtaina free DNA transferred cell. The cell can be then cultured and thecultured cell can be subjected to lineage study. Furthermore, it can beused for identifying a cell producing the oncogenic protein of thepresent invention and investigating association with apotosis,differentiation or growth or a signal transfer mechanism in them andabnormality in them. Thus, it can be an effective research subject forelucidating the oncogenic protein of the present invention and itsactivity.

Furthermore, in order to develop a therapeutic agent for a diseaseassociated with an oncogenic protein of the present invention such as adeactivation type refractoriness of the protein of the presentinvention, a DNA transferred animal of the present invention can be usedto provide an effective and speedy screening method for the therapeuticagent using the above test and assay processes.

A non-human mammal germinal stem cell in which a gene DNA homologue toan oncogene of the present invention is inactivated, is very useful forproducing a non-human mammal model insufficiently expressing theoncogene DNA of the present invention. A non-human mammal insufficientlyexpressing a gene DNA homologue to the oncogene of the present inventiondoes not have various biological activities which can be induced by theoncogenic protein of the present invention. It can be, therefore, usedas a model for a disease caused by inactivation of biological activitiesof the oncogenic protein of the present invention. Thus, it can beuseful for elucidating the cause of the disease and investigating atherapy therefor.

(8a) Method for Screening a Therapeutic or Prophylactic Compound to aDisease Caused by Deletion or Damage in an Oncogene DNA of the PresentInvention

A non-human mammal insufficiently expressing a DNA of the presentinvention can be used for screening a therapeutic or prophylacticcompound to a disease caused by deletion or damage in the DNA of thepresent invention.

Thus, the present invention provides a method for screening atherapeutic or prophylactic compound or its salt to a disease caused bydeletion or damage in a DNA of the present invention, comprising thesteps of administering a test compound to a non-human mammalinsufficiently expressing the DNA of the present invention and thenobserving and/or determining change in the animal.

A non-human mammal insufficiently expressing the DNA of the presentinvention used in the screening process may be as described above.

Examples of a test compound include a peptide, a protein, a non-peptidecompound, a synthetic compound, a fermentation product, a cell extract,a plant extract, an animal tissue extract and plasma, which may be anovel or known compound.

(8b) Method for Screening a Compound Promoting or Inhibiting a PromoterActivity to an Oncogene DNA of the Present Invention

The present invention provides a method for screening a compound or itssalt promoting or inhibiting a promote activity to an oncogene DNA ofthe present invention, comprising the steps of administering a testcompound to a non-human mammal insufficiently expressing the DNA of thepresent invention and then detecting expression of a reporter gene.

In the screening method, a non-human mammal insufficiently expressingthe DNA of the present invention may be, among the non-human mammalsinsufficiently expressing the DNA of the present invention, an animal inwhich the DNA of the present invention is inactivated by transducing areporter gene and the reporter gene can be expressed under the controlof a promoter to the DNA of the present invention.

Examples of a test compound include a peptide, a protein, a non-peptidecompound, a synthetic compound, a fermentation product, a cell extract,a plant extract, an animal tissue extract and plasma, which may be anovel or known compound.

A reporter gene which can be used may be as described above; suitablyβ-galactosidase gene (lacZ), soluble alkaline phosphatase gene andluciferase gene.

In the non-human mammal insufficiently expressing the DNA of the presentinvention in which the oncogene DNA of the present invention is replacedwith the reporter gene, the reporter gene exists under the control ofthe promoter to the DNA of the present invention, so that a substanceencoded by the reporter gene can be traced to detect an activity of thepromoter.

For example, when a part of a DNA domain encoding an oncogenic proteinof the present invention is substituted with an E. coli derivedβ-galactosidase gene (lacZ), β-galactosidase is expressed in place ofthe oncogenic protein of the present invention in a tissue in which theoncogenic protein of the present invention is naturally to be expressed.Thus, for example, by staining with a reagent to be a substrate forβ-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside(X-gal), an expression state of the oncogenic protein of the presentinvention in a living animal can be conveniently observed. Specifically,a mouse defective in a homologue protein to an oncogenic protein of thepresent invention or its tissue section is fixed by, for example,glutaraldehyde, washed with a phosphate-buffered saline (PBS) andreacted with a stain solution containing X-gal at room temperature orabout 37° C. for about 30 min to 1 hour. The tissue preparation thusobtained is washed with a 1 mM EDTA/PBS solution to quench theβ-galactosidase reaction and then a color can be observed.Alternatively, an mRNA encoding lacZ may be detected as usual.

A compound or its salt obtained using the above screening method isselected from the above test compounds, which can promote or inhibit apromoter activity to an oncogene DNA of the present invention.

The compound selected by the above screening method can form a salt,which may be a salt with a physiologically acceptable acid (for example,an inorganic acid) or base (for example, an organic base), preferably anacid-addition salt. Examples of such a salt include salts with aninorganic acid such as hydrochloric acid, phosphoric acid, hydrobromicacid and sulfuric acid, or with an organic acid such as acetic acid,formic acid, propionic acid, fumaric acid, maleic acid, succinic acid,tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid and benzenesulfonic acid.

Since a compound or its salt promoting a promoter activity to anoncogene DNA of the present invention promotes the activity of thepeptide, it is useful as, for example, a medical drug such as aprophylactic and a therapeutic agents for a disease associated withinsufficiency of an oncogenic protein of the present invention.

On the other hand, a compound or its salt inhibiting a promoter activityto a DNA encoding an oncogenic protein of the present invention isuseful as a safe and low-toxic medical drug inhibiting cancer-inducingactivity of the oncogenic protein of the present invention, for examplea prophylactic or therapeutic agent for a cancer such as pulmonarycancer, renal cancer, hepatic carcinoma, non-small cellpulmonary cancer,ovarian cancer, prostatic cancer, gastric cancer, bladder cancer, breastcancer, cervical cancer, colonical cancer, rectal cancer and pancreaticcancer.

A derivative of the compound selected by the above screening may be alsoused.

A drug comprising the compound or its salt selected by the screeningmethod can be prepared in a dosage form suitable for delivery to atarget cancerous tissue as in a drug comprising a known anticancer drugto a given cancer.

Since the formulation thus prepared is safe and low toxic, it can beadministered to a human as a main subject or a mammal in which a similarpharmacological effect would be expected (for example, rat, mouse,guinea pig, rabbit, sheep, pig, bovine, horse, cat, dog and monkey).

A dose of the compound or its salt depends on various factors such as atarget disease, a recipient and a delivery route. For example, in oraladministration of a compound inhibiting a promoter activity to a DNA ofthe present invention for treating a cancer, its dose is about 0.1 to100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20mg per day to a normal adult patient (weight: 60 kg). In parenteraladministration, although a dose of the compound depends on variousfactors such as a recipient and a target disease, its dose is suitablyabout 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferablyabout 0.1 to 10 mg per day by intravenous injection when a compoundinhibiting a promoter activity to a DNA of the present invention isadministered to a normal adult patient (weight: 60 kg) for treating acancer. For another animal, an amount converted to a weight of 60 kg maybe administered.

Thus, a non-human mammal insufficiently expressing an oncogene DNA ofthe present invention is considerably useful for screening a compound orits salt promoting or inhibiting a promoter activity to the oncogene DNAof the present invention, and therefore, can significantly contribute toelucidating causes of a variety of diseases due to insufficientexpression of the oncogene DNA of the present invention and developingprophylactic or therapeutic agents for the diseases.

Furthermore, using a DNA containing a promoter region for an oncogenicprotein of the present invention, a gene encoding a given protein can beligated to a downstream site, and the product can be injected in ananimal egg cell to produce a so-called transgenic animal (anintrogressant animal), so that a mechanism specifically causing in vivoexpression of the oncogene while avoiding cancerization. Furthermore, aproper reporter gene can be ligated to the promoter region to establisha transformant cell strain expressing the gene, which can be used as anin vitro search system for a low molecular-weight compound specificallypromoting or inhibiting an in vivo production ability of the oncogenicprotein of the present invention itself.

When being used for prevention or treatment of a cancer, a compound orits salt regulating activity of an oncogenic protein of the presentinvention can be combined with another anticancer agent such asifosfamide, UTF, adriamycin, peplomycin, cisplatin, cyclophosphamide,5-FU, UFT, methotrexate, mitomycin C and mitoxantrone.

EXAMPLES

The present invention will be more specifically with reference toExamples, These examples are included in the most preferred embodimentsof the present invention, but the present invention is not limited tothese embodiments.

Example 1 Cloning and Sequencing of a cDNA Encoding a hWAPL from Human

PCR was conducted using a commercially available cDNA library, a humantesticular cDNA kit (Marathon-Ready™ cDNA Kit; Clontech Inc.) as atemplate and using two primers:

primer 1 (sequence: TTGGATCCATGACATCCAGATTTGGGAAAACATACAGTAGG) (SEQ IDNO: 8); and

primer 2 (sequence: TTGAATTCCTAGCAATGTTCCAAATATTCAATCACTCTAGA) (SEQ IDNO: 9). In the PCR reaction, Advantage 2 polymerase mix (Clontech Inc.)kit was used and according to the instructions in the kit, amplificationwas conducted with a temperature cycle:

(1) 94° C. for 1 min;

(2) 5 cycles of 94° C. for 10 sec and 72° C. for 2 min; and

(3) 25 cycles of 96° C. for 10 sec and 70° C. for 2 min, and thenfollowed by elongation reaction being carried out at 72° C. for 5 min.After the reaction, the product of PCR amplification thus obtained wascloned to a plasmid vector pGEM-T easy (PROMEGA) in accordance with theinstructions for pGEM-T easy (PROMEGA). It was transduced to E. coli DH5α (Invitrogen) and using an ampicillin resistance gene in the plasmidvector pGEM-T easy, clones carrying the plasmid were selected in an LBagar medium containing ampicillin.

The plasmid carried by each of the selected clones was sequenced toobtain the sequence of the novel cDNA (SEQ. ID. No.2) cloned therein. Anovel protein comprising an amino acid sequence (SEQ. ID. No.1) deducedfrom an ORF in the nucleotide sequence of the cDNA was designated as ahuman WAPL (hWAPL). A transformant carrying the full-length cDNA of thehWAPL, which was cloned in the plasmid, was designated as Escherichiacoli DH5 pGEMhWAPL.

Example 2 Expression of an hWAPL Gene in a Human Cancerous Tissue

Under the consent of patients subjected to surgical excision of acancerous tissue in Tokyo Medical College Hospital, the samples ofsurgically excised cancerous tissues were provided. The samples ofsurgically excised cancerous tissues were examined for the presence ofhWAPL gene expression and the expression amount thereof by Northernblotting and real-time PCR.

In the Northern blotting, an mRNA expressed from the hWAPL gene wasidentified from total RNAs prepared, using a DNA having a nucleotidesequence complementary to the portion of nucleic acid Nos. 2511 to 2813in the full-length sequence of the hWAPL of SEQ. ID. No.2, as adetection probe. On the other hand, in the real-time PCR, a cDNA of thehWAPL was amplified, using an amplification kit SYBR Green I (TaKaRa Co.Ltd.) and as PCR primers, a pair of primers:

5′-GAATTCATAGGCACAGCGCTGAACTGTGTG-3′ (SEQ ID NO: 5) and

5′-TTGAATTCCTAGCAATGTTCCAAATATTCA-3′ (SEQ ID NO: 6).

Furthermore, human β-actin was used as an intrinsic standard. PCRprimers used for amplification of cDNA of human β-actin were acommercially available pair of primers (Clontech) of:

5′-GGGAAATCGTGCGTGACATTAAG-3′ (SEQ ID NO: 10) and

5′-TGTGTTGGCGTACAGGTCTTTG-3′ (SEQ ID NO: 11).

The temperature cycle condition selected for the real-time PCR was asfollows:

(a) 95° C. for 30 sec;

(b) 40 cycles of 95° C. for 3 sec and 68° C. for 30 sec; and then

(c) 87° C. for 6 sec.

For the thermal cycle in the PCR reaction, Smart Cycler System (TaKaRaCo. Ltd.) was used. The amount of the double strand cDNA was detected bymeans of a fluorescent label therein to determine the amount ofamplification product.

For the cancerous tissue samples mentioned above, comparative samples oftotal RNA were prepared from normal cells and carcinoma cells in theindividual tissues in accordance with a known procedure (Oikawa et al.,Cancer Res., 61, 5707-5709 (2001)). Among the carcinoma cells examinedof cervical cancer, corpus uteri cancer, ovarian cancer, gastric cancer,renal cancer, pulmonary cancer, colorectal carcinoma and breast cancer,about 40% of carcinoma cell samples of cervical cancer demonstratedsignificant hWAPL gene expression (FIG. 5).

The carcinoma cell samples of cervical cancer were examined for thepresence of HPV infection. Specifically, an mRNA region for theHPV-derived E6/E7 gene was detected by RT-PCR with corresponding primerstherefor in accordance with a known procedure (Nakagawa et al., J. Med.Virol., 62, 251-258 (2000)), and in all the invasive carcinoma cellsamples of cervical cancer, HPV infection and expression of the E6/E7gene were confirmed. Exceptionally, some of gastric cancer samples alsodemonstrated high expression of the hWAPL gene.

Example 3 Induction of hWAPL Gene Expression by E6 and E7 from HPV 16

An E6 gene from HPV 16 was amplified and isolated by RCR method using apair of primers

16E6attB1:

5′-AAAAAGCAGGCTCCACCATGTTTCAGGACCCACAGGAGCGACCC-3′ (SEQ ID NO: 12), and

16E6attB2:

5′-AGAAAGCTGGGTTACAGCTGGGTTTCTCTACGTG-3′ (SEQ ID NO: 13),

while an E7 gene from HPV 16 was amplified and isolated by RCR methodusing a pair of primers

16E7attB1:

5′-AAAAAGCAGGCTCCACCATGCATGGAGATACACCTACAT-3′ (SEQ ID NO: 14) and

16E7attB2:

5′-AGAAAGCTGGGTTATGGTTTCTGAGAACAGATGGGG-3′ (SEQ ID NO: 15).

Then, each of these genes were cloned into a retrovirus vector pCLXSN inaccordance with a procedure of Naviaux et al. (Naviaux et al., J.Virol., 70, 5701-5705 (1996)), to generate retroviruses LXSN-16E6 andLXSN-16E7 for producing E6 and E7 recombinants, respectively. Inaddition, the retrovirus LXSN-16E6E7 for producing E6 and E7recombinants simultaneously was produced.

It has been found that an product of E2 gene derived from HPV can bebound to a promoter region in the E6 and E7 genes to possess a functionfor inhibition of the transcription thereof, and that a product of E2gene derived from a bovine papilloma virus (BPV) has a similar functionfor inhibiting the transcription. The BPV1 E2 gene fragment was obtainedby nested PCR using pBPV-MII as a template. In the nested PCR, the pairof inner primers used was consisted of:

5′-AAAAAGCAGGCTCCACCATGGAGACAGCATGCGAAC-3′ (SEQ ID NO: 16) and

5′-AGAAAGCTGGGTCAGAAGTCCAAGCTGGCTGTAAAG-3′ (SEQ ID NO: 17),

while the pair of outer primers was consisted of:

5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3′ (SEQ ID NO: 18) and

5′-GGGGACAAGTTTGTACAAGAAAGCTGGGT-3′ (SEQ ID NO: 19).

The BPV1 E2 gene fragment thus obtained was cloned into a retrovirusvector pCMSVpuro, which is based on a general-purpose virus vectorpCMSCV (Clontech), to prepare a retrovirus MSCV-puro BPV1E2 forproducing the E2 recombinant from BPV. The retrovirus vector pCMSVpurocomprises a puromycin resistance gene as a selection marker.

Human epidermal cells HDK1 (BioWhittaker) were infected with theretroviruses LXSN-16E6, LXSN-16E7 and LXSN-16E6E7 for producing HPV 16derived E6 or E7 recombinants, respectively. As a negative control, ahuman epidermal cell HDK1 infected with a retrovirus vector pCLXSN wasused. A cell line in which continued infection of the retrovirus vectorwas established was selected by culturing the cells on a mediumcontaining G418 at 50 μg/mL for 3 days. After infection, expression ofthe hWAPL gene induced by a recombinant protein of the E6 or E7 from HPV16 was determined by Western blotting using a specific antibodyrecognizing a region of partial amino acid sequence 50 to 66 (amino acidsequence: CNFKPDIQEIPKKPKVEE (SEQ ID NO: 20)) in the oncogenic proteinhWAPL (FIG. 6).

At the same time, the amount of expression of p53 suppressor protein wasalso determined by Western blotting. Then, it was again confirmed thatE6 product promoted a cleavage process of the p53 tumor suppressorprotein, resulting in expression of the oncogenic protein hWAPL.

Furthermore, when the carcinoma cell lines of cervical cancer; CaSki,SiHa and C33A were infected with MSCV-puroBPV1E2, CaSki and SiHa celllines producing E6 and E7 from HPV 16 demonstrated increase of theremained p53 tumor suppressor protein as a result of inhibitingtranscription of E6 and E7 genes by the E2 from BPV. Concomitantly,expression of the oncogenic protein hWAPL was inhibited (FIG. 6). On theother hand, in the C33A cell line, in which cancerization was induced bymechanism other than such cancerization being originated by HPVinfection, the amount of the remaining p53 tumor suppressor protein orthe amount of expression of the oncogenic protein hWAPL was notaffected. The results indicate that although the increased amount ofexpression of the oncogenic protein hWAPL is closely associated withcancerization, an independent mechanism causing increased expression ofthe oncogenic protein hWAPL may exist, which is different from themechanism of increased expression of the oncogenic protein hWAPL due toE6 and E7 from HPV.

Example 4 Inhibition of Promoter Activity of the hWAPL Gene by a p53Suppressor Protein

It was confirmed that the p53 suppressor protein has a function forinhibition of transcription of the hWAPL gene from its promoter, asfollows.

A promoter of hWAPL gene was amplified and isolated by PCR method usinga genomic DNA in DLD-1 cell as a template with use of a pair of primers:

primer 1

(sequence: GTGCATCCCACCCACAGTGGAAGACATGG) (SEQ ID NO: 21) and

primer 2

(sequence: CCGCTTCCGCCGGTGAATGGTCAGTGCTGG) (SEQ ID NO: 22).

A DNA fragment of the PCR product was first cloned to PGEMT-easy(PROMEGA), and sequentially, the fragment digested with a restrictionenzyme EcoRI was then cloned to pBluescript (Stratagene). A regioncomprising a promoter portion of the hWAPL gene, which was inserted intothe plasmid, was digested with SalI/XhoI and cloned into the pGL3-Basicvector (PROMEGA).

After purification with Qiagen Plasmid Maxi Kit (Qiagen), the vectorobtained was co-transfected with a p53 expression vector, pHM6 (Roche)as for a control and a pGR3-tK vector for standardization of luciferaseassay, using LipofectAmine2000 (Invitrogen) in an HeLa cell. DualLuciferase Kit (Promega) was used in the luciferase assay fordetermining the amount of labeled protein, luciferase, which was areporter gene product transcribed and translated under control of thepromoter of hWAPL gene.

As a result, it was confirmed that when transducing the p53 expressionvector, increase in the amount of the expressed p53 suppressor proteinreduced the amount of the labeled protein luciferase, which wastranscribed and translated under control of the promoter of hWAPL gene,by 30% in comparison with the control. In other words, it is confirmedthat the p53 suppressor protein has a function causing reduction inactivity of the promoter of the hWAPL gene.

Example 5 Construction of an Expression Vector for Producing the hWAPLRecombinant Protein

A HindIII and EcoRI restriction sites was transduced respectively at theends of the region of base Nos. 1 to 3570 in the cDNA having thenucleotide sequence of SEQ. ID. No.2 by site-specific mutagenesis usingPCR. The PCR product thus obtained was digested with HindIII/EcoRI, andthe corresponding DNA fragment was inserted into an HA-tagged mammalianexpression vector, pHM6 (Roche Diagnostics) to construct an expressionvector for producing a recombinant of the HA-tagged hWAPL protein,pHM6-hWAPL. The fragment was also inserted into an mammalian expressionvector, phrGFP-N1 (Stratagene) for expressing a fused protein with anhrGFP fusion partner to construct an expression vector for producing arecombinant protein of a GFP fused hWAPL, phrGFP-hWAPL.

The expression vector for producing a recombinant was transfected to ahost mammal cell using LipofectAmine 2000 (Invitrogen). After culturingfor 2 days following the transfection, selection is conducted usingEPICS ALYRA HyperSort (Bechman Coulter) on the basis of the presence ofexpression of the hrGFP tag. After further culturing and additionalselection, a transformed host cell line carrying the desired expressionvector for producing the hWAPL recombinant protein.

Example 6 Generation of an Antibody Specific to the hWAPL Protein

For generating an antibody specific to the oncogenic protein hWAPL, apeptide chain (hWAPL₅₀₋₆₆) having a partial amino acid sequence 50 to66: CNFKPDIQEIPKKPKVEE (SEQ ID NO: 20) that is located in the N-terminalregion of the oncogenic protein hWAPL was prepared as an immunogenpeptide by chemical synthesis. Furthermore, by recombination wasproduced a fused polypeptide tagged with 6×H His comprising a partialamino acid sequence 814-1037 lying in the C-terminal region of theoncogenic protein hWAPL.

According to conventional technique, these two immunogen peptides wereused to separately immunize a rabbit to produce polyclonal antibodiesspecific to these antigen peptides, an anti-hWAPL-N antibody and ananti-hWAPL-C antibody. In Western blotting in Example 3 and so on, theoncogenic protein hWAPL was detected using the anti-hWAPL-N antibodyspecific to the hWAPL₅₀₋₆₆ antigen.

Example 7 Induction of Chromosome Instability by the hWAPL Protein

A HeLa cell is infected with the expression vector phrGFP-hWAPL forproducing a recombinant protein of the hrGFP-fused hWAPL. The proceduredescribed in Example 5 is conducted to select a GFP-hWAPL positive cellline producing the GFP-hWAPL-fused protein and a GFP-hWAPL negative cellline not producing the GFP-hWAPL-fused protein. After subculturing forfive generations following the selection, the content of a chromosomegene DNA therein is assayed by Laser Scanning Cytomerty in accordancewith Buse's procedure (J. Biol. Chem., 274, 7253-7263 (1999)).

The GFP-hWAPL negative cell line demonstrates a chromosome gene contentequivalent to the host cell, HeLa cell, in which a half or more iscomprised of a normal diploid and a content of a tetraploid is about ahalf of the diploid. On the other hand, in the GFP-hWAPL positive cellline, a normal diploid exists in a less amount, a major partdemonstrates polyploidy, a content of a tetraploid is slightly more thanthat of a diploid, and an octoploid also exists at a content of 10.1%.Thus, it can be judged that over-expression of the hWAPL protein induceschromosome instability (FIG. 7).

Correspondingly, induction of nuclear atypia, in particular, ofmulti-nucleation is also originated from over-expression of the hWAPLprotein. After three generation subculturing, ratios of observedmulti-nucleation are only 5% and 4% in the GFP-hWAPL negative cell lineand the HeLa cell infected with the phrGFP-N1 vector (control),respectively, while being 15.6% (three times or more) in the GFP-hWAPLpositive cell line (FIG. 8).

Furthermore, in association with chromosomal abnormality such as nuclearatypia due to abnormal centromere division during a separation processby mitosis of a chromosome gene, induction of micronuclei formation isoriginated by over-expression of the hWAPL protein. A frequency ofmicronuclei formation in the GFP-hWAPL positive cell line is about twotimes as much as that in the GFP-hWAPL negative cell line (FIG. 7).

Example 8 Induction of Cancerization of an NIH 3T3 Fibroblast by thehWAPL Protein

An NIH 3T3 fibroblast is infected with the expression vector pHM6-hWAPLfor producing a recombinant protein of the HA-tagged hWAPL to produce arecombinant cell line over-expressing the HA-tagged hWAPL protein, anHA-hWAPL 3T3 cell line. As a negative control, an HA-3T3 cell lineinfected with a pHM6 vector is produced. When culturing on a plate, theHA-3T3 cell line for negative control forms a homogenous confluent-likesingle cell layer as in the host cell, NIH 3T3 fibroblast, while theHA-hWAPL 3T3 cell line forms a focus structure.

To a nude mouse was subcutaneously injected the HA-hWAPL 3T3 cell lineand the HA-3T3 cell line. Subsequent follow-up indicated that within 10days, oncogenesis was induced in all the injection sites for theHA-hWAPL 3T3 cell line while no oncogenesis was induced in injectionsites for the HA-3T3 cell line. By Western blotting using the anti-HAantibody and the anti-hWAPL-C antibody, it was confirmed that theHA-tagged hWAPL protein was actually over-expressed in the cancerizedcell. In the cancerized cell, heterotypic mitosis was observed; forexample, three pole division was also observed.

Example 9 Inhibition of Carcinoma Cell Growth by an siRNA Targeted tothe hWAPL Gene

Using an siRNA targeted to the hWAPL gene, it was confirmed thatinhibiting expression of the hWAPL protein can result in inhibition ofgrowth of a carcinoma cell.

(1) In Vitro Inhibition of Carcinoma Cell Growth by an siRNA

Using the Silencer siRNA construction Kit (Ambion), siRNA were producedwhich are targeted to the following gene sequence (hWAPL AsiRNA) and toa control (negative control), respectively:

hWAPL AsiRNA: CGGACTACCCTTAGCACAA (SEQ ID NO: 7)

negative control: ACTACAACTGGTCGCAACC (SEQ ID NO: 23).

Practically, two synthetic oligomers were prepared for each;specifically, for the hWAPL AsiRNA,

AACGGACTACCCTTAGCACAAcctgtctc (SEQ ID NO: 24) and

AATTGTGCTAAGGGTAGTCCGcctgtctc (SEQ ID NO: 25),

and, for the negative control,

AAACTACAACTGGTCGCAACCcctgtctc (SEQ ID NO: 26) and

AAGGTTGCGACCAGTTGTAGTcctgtctc (SEQ ID NO: 27).

Then, in each synthetic oligomer, a T7 promoter primer was hybridized atportion of ctgtctc and was treated with Klenow DNA polymerase to preparecompletely a double strand DNA. Then, it was transcribed by T7 RNApolymerase, the RNAs of the antisense and sense chains prepared werehybridized each other, and then both cohesive ends were digested withRnase to prepare a double strand siRNA with blunt ends.

To a SiHa cell derived from cervical cancer, in which hWAPL was highlyexpressing, was transduced the hWAPL AsiRNA and the negative controlsiRNA at a concentration of 1 nM for evaluating influence to cellgrowth.

The siRNAs were transfected to the SiHa cell derived from an HPV16positive cervical cancer, in which high expression of the hWAPL wasobserved. FIG. 10 shows the results of plotting the living cell number(×10³) in the ordinate to a time from transduction of the siRNA (hour)in the abscissa. In the figure, “siRNA” for transfection of an siRNAtargeting DIF-2, “cont” for transfection of a control siRNA, and “TSA”indicate the results obtained when adding a histone deacetylaseinhibitor, Trichostatin A to a culture after transfection of the siRNA,respectively.

Until 20 hours after transfection of the hWAPL AsiRNA, increase in theliving tumor cell number can be observed. During the period, comparisonwith the control may indicate inhibition of tumor growth. Subsequently,the cells to which the hWAPL AsiRNA was transduced show reduction in theliving cell number and after 100 hours, a small number of cells wereliving. In contrast, when progress of deacetylation for acetylatedlysines contained in a histon is inhibited by adding Trichostatin A, itattenuates effect of inhibition of cell growth by the hWAPL AsiRNA.

These results indicate that cell death by inhibition of an hWAPL iscaused by inhibition of histon modification such as acetylation, andimplies that the hWAPL itself may be involved in control of a histoncode.

(2) In Vivo Inhibition Effect of Tumor Growth

To six-week old BALB3T3/nude mice were inoculated 2×10⁶ of SiHa cells.From 10 days after inoculation, to the inoculated tumor cells wereinjected hWAPL AsiRNA and a negative control siRNA once two days andfive times in total, respectively.

Variation of the inoculated tumor size was evaluated for 6 animals inthe hWAPL AsiRNA injection group, 6 animals in the negative controlsiRNA injection group and 5 animals in the untreated group. The resultsindicated an average tumor size reduction of 33% in the hWAPL AsiRNAinjection group in relation to the untreated group.

Furthermore, it was demonstrated that the nucleotide sequence of thecDNA for the hWAPL, which we cloned, was located in 10q23.31 to q23.32on the human genome. The nucleotide sequence thereof is shown below,along with the nucleotide sequence of the 5′-untranslation region andpromoter region in the elucidated hWAPL gene as well as the nucleotidesequence of cDNA for the above mouse WAPL. In the ORFs in the cDNAs, aregion from the initiating codon ATG to the stop codon TAG is written bycapitals, while in the promoter region, a region after a putativetranscription initiating point is written by capitals on the basis ofcomparison with EST.

cDNA Sequence of the hWAPL (SEQ ID NO: 28)

gcgagcggctgttggaggaaggaggtgggggccgggagcgcaaatggcgttgagatggtycarggccctgttcaaactccagcactgaccattcaccggcggaagcggcggcgcaggaggcggcggcggcccagcgggggcacacagcaggctctgttaccagctccagcagtggcggccagcgagagctaggcccgsgcccggccggcggcgctcgaggcggggagggaagttgcggggccgccgctcctgcccccccaaccgggcttcctatttaccgaaagcagagtccctcgcctctctcggctctcacctgccggccctgctctcccgcgcgagggttccgcgcccgcccgcgggccgtarggagcgggagaaggcggargcggccccgtggccaaagcacccgccaggcttccgaggagaatatgaaactggtgtcaaaATGACATCCAGATTTGGGAAAACATACAGTAGGAAAGGTGGAAATGGCAGTTCAAAATTCGATGAAGTCTTTTCCAACAAACGGACTACCCTTAGCACAAAATGGGGAGAGACCACATTTATGGCTAAATTAGGGCAGAAGAGGCCCAATTTCAAACCAGATATCCAAGAAATTCCGAAGAAACCTAAAGTGGAAGAAGAAAGTACTGGAGATCCTTTTGGATTTGATAGTGATGATGAGTCTCTACCAGTTTCTTCAAAGAATTTAGCCCAGGTTAAGTGTTCCTCTTATTCAGAATCTAGTGAAGCTGCTCAGTTGGAAGAGGTCACTTCAGTACTTGAAGCTAATAGCAAAATTAGTCATGTGGTCGTTGAAGACACTGTCGTTTCTGATAAATGCTTCCCTTTGGAGGACACTTTACTTGGGAAAGAAAAGAGCACAAACCGAATTGTAGAAGATGATGCAAGCATAAGTAGCTGTAATAAATTAATAACTTCAGATAAAGTGGAGAATTTTCATGAAGAACATGAAAAGAATAGTCACCATATTCACAAAAATGCTGATGACAGTACTAAGAAACCCAATGCAGAAACTACAGTGGCTTCTGAAATCAAGGAAACAAATGATACTTGGAACTCCCAGTTTGGGAAAAGGCCAGAATCACCATCAGAAATATCTCCAATCAAGGGATCTGTTAGAACTGGTTTGTTTGAATGGGATAATGATTTTGAAGATATCAGATCAGAAGACTGTATTTTAAGTTTGGATAGTGATCCCCTTTTGGAGATGAAGGATGACGATTTTAAAAATCGATTGGAAAATCTGAATGAAGCCATTGAGGAAGATATTGTACAAAGTGTTCTTAGGCCAACCAACTGTAGGACGTACTGTAGGGCCAATAAAACGAAATCCTCCCAAGGAGCATCAAATTTTGATAAGCTGATGGACGGCACCAGTCAGGCCTTAGCCAAAGCAAACAGTGAATCGAGTAAAGATGGCCTGAATCAGGCAAAGAAAGGGGGTGTAAGTTGTGGGACCAGTTTTAGAGGGACAGTTGGACGGACTAGAGATTACACTGTTTTACATCCATCTTGCTTGTCAGTTTGTAATGTTACCATACAGGATACTATGGAACGCAGCATGGATGAGTTCACTGCATCCACTCCTGCAGATTTGGGAGAAGCTGGTCGTCTCAGAAAAAAGGCAGATATTGCAACTTCTAAGACTACTACTAGATTTCGACCTAGTAATACTAAATCCAAAAAGGATGTTAAACTTGAATTTTTTGGTTTTGAAGATCATGAGACAGGAGGTGATGAAGGAGGTTCTGGAAGTTCTAATTACAAAATTAAGTATTTTGGCTTTGATGATCTCAGTGAAAGCGAAGATGATGAAGATGATGACTGTCAAGTAGAAAGAAAGACAAGCAAAAAAAGAACTAAAACAGCTCCATCACCCTCCTTGCAGCCTCCCCCAGAAAGCAATGATAATTCCCAGGACAGTCAGTCTGGTACTAACAATGCAGAAAACTTGGATTTTACAGAGGACTTGCCTGGTGTGCCTGAAAGTGTGAAGAAGCCCATAAATAAACAAGGAGATAAATCAAAGGAAAATACCAGAAAGATTTTTAGTGGCCCCAAACGGTCACCCACAAAAGCTGTATATAATGCCAGACATTGGAATCATCCAGATTCAGAAGAACTGCCTGGGCCACCAGTAGTAAAACCTCAGAGTGTCACAGTGAGGCTGTCTTCAAAGGAACCAAATCAAAAAGATGATGGAGTTTTTAAGGCTCCTGCACCACCATCCAAAGTGATAAAAACTGTGACAATACCTACTCAGCCCTACCAAGATATAGTTACTGCACTGAAATGCAGACGAGAAGACAAAGAATTATATACTGTTGTTCAGCACGTGAAGCACTTCAACGATGTTGTAGAATTTGGTGAAAATCAAGAGTTCACTGATGACATTGAGTACTTGTTAAGTGGCTTAAAGAGCACTCAGCCTCTAAACACACGTTGCCTTAGTGTTATTAGCTTGGCTACTAAATGTGCCATGCCCAGTTTTCGAATGCACCTGAGAGCACATGGGATGGTAGCAATGGTCTTTAAAACCTTGGATGATTCCCAGCACCATCAGAATCTGTCCCTCTGTACAGCTGCCCTCATGTATATACTGAGTAGAGATCGTTTGAACATGGATCTTGATAGAGCTAGCTTAGATCTAATGATTCGACTTTTGGAACTGGAACAAGATGCTTCATCAGCCAAGCTACTGAATGAAAAAGACATGAACAAAATTAAAGAAAAAATCCGAAGGCTCTGTGAAACTGTACACAACAAGCATCTTGATCTAGAAAATATAACGACTGGGCATTTAGCTATGGAGACATTATTATCCCTTACTTCTAAACGAGCAGGAGACTGGTTTAAAGAAGAACTCCGGCTTTTGGGTGGTCTGGATCATATTGTAGATAAAGTAAAAGAATGTGTGGATCATTTAAGTAGAGATGAGGATGAAGAGAAACTGGTAGCCTCACTATGGGGAGCAGAGAGATGTTTACGAGTTTTAGAAAGTGTAACTGTGCATAATCCCGAAAATCAAAGCTACTTGATAGCATATAAAGATTCCCAACTTATTGTTTCATCAGCTAAAGCATTACAGCATTGTGAAGAACTGATTCAGCAGTACAACCGTGCTGAGGACAGCATATGCTTAGCTGACAGTAAGCCTCTGCCTCACCAGAATGTAACTAACCATGTAGGCAAAGCAGTGGAGGACTGCATGAGGGCCATCATCGGGGTGTTGCTTAATTTAACTAATGATAATGAGTGGGGCAGCACCAAAACAGGAGAGCAGGACGGTCTCATAGGCACAGCGCTGAACTGTGTGCTTCAGGTTCCAAAGTACCTACCTCAGGAGCAGAGATTTGATATTCGAGTGCTGGGCTTAGGTCTGCTGATAAATCTAGTGGAGTATAGTGCTCGGAATCGGCACTGTCTTGTCAACATGGAAACATCGTGCTCTTTTGATTCTTCCATCTGTAGTGGAGAAGGGGATGATAGTTTAAGGATAGGTGGACAAGTTCATGCTGTCCAGGCTTTAGTGCAGCTATTCCTTGAGCGAGAGCGGGCAGCCCAGCTAGCAGAAAGTAAAACAGATGAGTTGATCAAAGATGCTCCCACCACTCAGCATGATAAGAGTGGAGAGTGGCAAGAAACAAGTGGAGAAATACAGTGGGTGTCAACTGAAAAGACTGATGGTACAGAAGAGAAACATAAGAAGGAGGAGGAGGATGAAGAACTTGACCTCAATAAAGCCCTTCAGCATGCCGGCAAACACATGGAGGATTGCATTGTGGCCTCCTACACGGCACTACTTCTTGGGTGTCTCTGCCAGGAAAGTCCAATCAATGTAACCACTGTGCGGGAATATCTGCCAGAAGGAGACTTTTCAATAATGACAGAGATGCTCAAAAAATTTTTGAGTTTTATGAATCTCACTTGTGCTGTTGGAACAACTGGCCAGAAATCTATCTCTAGAGTGATTGAATATTTGGAACATTGCTAGctgctttacctttgcttcaggtgctcggtaatgctggagctatccttagacaaagaaaagtcaagtcatgaaagaagtccttgaagatataccaagaacattcatcagtatcattcgtgtttggatttttaaggccacctgatttcttcgtcatgcattcggcatttgctaaatgacagttactacatcaatctgcaactatcaaaaatgaggggaaaaggttcaggctgttaacaattccatgcagtatttaaatacatttactttggcagagtttataccctccccttgttttcttgctttattctgggcaagtttgaaggggaaaatttgtgctgctgttagtgcaactgctgtgtatgttgagccactgttgtcatgccagccaggtgcaaaggcagcttagctactgaggtagcgaatgttctgaggacattctagacaacagcttagttcctttttcaggctcatttgcttttgcttttttgttgaatgattccaatcgtaaataaagcttttaataattttgtgaattttttggttgttgttccctgaactactgtctatatttaaaattagatggaatccaaagatacacgggattaatagtatatttttttattcttgattaggtttgggttattgaactattttttacttttgagaccacaaccatattcaatatcataccataatgtgtcatagctataggcacaagaaaaacaacagtttgagagaatattatataagatgatgtgccctgttaaaaggaggaggcaaaatagtcaaacccagggtagtttacacttaatgctagggaggctcttaaaacattattagattttgaggaaagactctctagatatattttctaatgttcagtacaataaatataaggaagctaaaacaccaatgtggaattcctgtttccagataacatgtatattcttctatagagtgacaggatcaattgcataagcgcaaagccttaaattgctggtttagagaagacccttttttcattcagattctttgttcgtagagcagttatttgaaaaacagttatggaacacaaaacattttatagatttaatatcataacattgcaaatttttcttgtattattgttcacaccactggttatacttttttttttccttttttattgattgggcctgaatacaggctttctagagatctttttcattaatacttttaaatacctttcaggtagttacatcatgtttcttcattggatttgtaaaacttgaagccataaaaatattagtttggtgtgtattggggaaaatagctaaaagtctaatttttacccatttagactttgttatttccttgtataaagtgacaaatcggggctcttgtatcagtgccagctgtaatgtttttaaatgcagtggctgccttctattgtcttcctatttttgataatgcagattgttgggaaatctgtaaggaagtaactgattccaggcaaattgttttcttccttctacccaccccaacccctacccatcaccttttaagaacatagtacgccagtgtaacgtgggaaccattgagattgtatttgccctgagtattaaagctagcttagcaaaatacttttaaaaacatattggtaaatgatacccataaaattaaattagttatattttattttaaaatgcaaaatacattgatatttattaatcattggatttagggaaagggacagatttttggtgaacctgacttgtggcagatggtaaggaatattataaaacatttggatgagaacaatcagggcgaactgcatttttctgttacactggtaatcatttgaaaattgatttacctcagtgtttaacagttttttgttttgttttgttttttaaataataactaattgtcgagcactgatagagatgcagattttggtggggggaggtggtgggggagataatcacttcaccaactgcagtgcatttgtgtgtttttaaccctcagagaactctgcattttagggtacttgaggctgacttaactaaaagttttaaagtaaccttttttccattgtaaatatttctgtaaatactaccaattggaaattagaacagtagagtacttttctgaatccaatcctatttttattttatacagtatttctcagctgtgatctttggagcaaaagccaacggcaggaaaaaatagtttgtaccagtttcatgaagtatgtctttgggtttttgtaaataattttaactcaaataaaattgctactttcaatacPromoter Region Sequence of the hWAPLgene (SEQ ID NO: 29)atttttagtagagacggggtttcaccgtgttagccaggatggtctcgatctcctgacctcatgatccgcctgcctcggcctctcaaagtgctaggattacaggcgtgagccaccgtgcctggccgctgaacacaatttaaagcttcaattaatccaggtattcagtcaacaaatatttatagcacactttctgtgtgtgaggcactattctaggtgtgcttggcatataaaatgaacaaaagtcagccatcctcgtcctcatggagtttatattcttgtgaaacgaaatagataataaacaagttcatacacaaagcaaacgtaatgactatgttatggagaaaagcagcagggaaaaggagatacagggtgctggatgaccttaaatagcatggccaaggaaaacattactgagagacacacttgagcaaagacctgaaagcatgcagggaatgagttgtgtgtgtctcttgaggactaacagagggaacaagtacgaagagggcccacaggcaggagctggcttggcatgttctagtagtagacagaggcaggcccggcaaggtaggaagggataggagtactcaggggccagatcatgcagggccttttcaccgttaagaactttggattttagtattacaggaggacccttcagggtgtttgactaggcgggtatcatacagtattaagggtgaggatcctgaataaaaaagggctgtttccaggacaagggtcaggaagccagacttcttcgaggttgcttgtaccggtccttgtcaggcaatgtgctcctagagaatttcctttgctttgtgtttcatctacctagacagcagtgtattccccagaggacgtcactatctccagagaacatattccaattatcctgggaaatatgataattgggattataacagtactcattttctcaattctcagaatgaaaacctatccaaggcaagaacaaaagttctccagaaagcactcccctcccaattgtgaaaacccagttaacattttattagagctaccaggttatgtgaaactgttgatagttttatcactttcctttcaagatataggcaggggcagtggctcatgcctgtaattccagcactttgggaggccaaggtaggtggatggcttgagcccagaagttcaagaccagcctgggcaacaaggtgaaaccctatctctaccaaaaatacaaaaattagtggggcatgatagcatggacatgtagtcccagctacttgggaggctgaggtgggaggatggcttgaaccctggaggtggaggttgcagtgattggagatcgtgccactacattccagcctgggcgacagagcaagactctgtcaccaaaaaaaaaaaaaaaaaaaaaaaaaaaaaatgtggccaggcatggtagctcacacctgtaatcccagcactttgggaggctgagccaggcggatcacaaggtcaagagatcgaaaccatcctggccaacatgcaaaacaccatctctactaaaatacaaaaaatgagctgggtgtggtggtgtgcgcctgtagtcccagctactggggaggctgaggcaggggaatcgcttgaaccagggaggcagagattgcagtgagccgagatcgcgccactgcactccagcctggtgacagagcgagactccgtctaaaaaaaaaaaaaaaaaaatatatatatatatatatataatatatgtatataattttacatgaaagaaggaaataaatgggtgcttttattcaacaaatatttattgagcacctactcttgtgccaggcagtcttctaggtgctagggttgcagcagaaaacaagacaggcagagatccctgccttcagagggagcacaacatttaagataaacatgcaaaatgcctgatatgttagatgggaagataaatgcccttaaagaaagtaaagcagggacagtgacatttaggagtgaggatgttgcaacaatttaagataggttggtcaggggagattcattgaaaagtcccatctgagtgaaaacctagaggagagaattgaagcaggctggtatctgggggagttgtaggcagagggaataggaaatacaaaggccctaaggtggggaaacagcaaggagtcaggtgtgggcagagcagaaagttggggctgcatgagcaaagggcagggcctggcccacatctcgtagagctttgtaatataaccacaacatgcaagtgtacaatttaacattttattccacatccgatggcaaagaaaaattgactgctaccaatatggtagtttctgacctgtagttccctaaatagaattctataagttgtaatacactttactacacattatcagaaaaagactaaaagttctatttagtaactccaattctgacagttctcatgtctgggctagaacaagggcatggcaatggcagaacagatgtcttctattttctttgcaggattttcttttttcagaggaaagtacaggtatgggcccactcaagtggagctccaggttagggggttctgtcctcacccaggcagcacacaggagggcagaggcccctcctaagggtactacaaacttggctctgatccatgatttcagtttctgacaaacacaacattcagtgggggaaagaaaatcaggtatctgagagcttgcacacaggcattctagcaaaaccaaaagcacctactggctacttgatgttagtgtgaagattctcatgaaatggagacaaccattctaggggttgaggtggccagggggatggagcctgagctgagagaactaagaaaacaaaaacaatacaacaaaaagctgttcagccatgtgttacccacactggagttctgtttgctcattctggtgtggaacccagggccctggtaagggaatgaggggacttcagggcatttgcttgcctcagtggaagcagggaggtaaggggttaaggtggtggtacagcctgcagggccaggagtctgaactccctccaggaggggcccgaggggtgtctttagtgtgagccacacaagggtacagagcccagaagagctctgcttaatattcatatactaagcttccacagactaaatacacacacacacacacacacacacacacactcacacacactttacttctcaaatcatgtaccactttctaccagattcaagaacaccaagaagtactaaagggtatccaaccgtcaagaaaaagtttgactccctcattagttgtaacatacaagtcttcccatttccttacttgtaagatagagtaatggactgggaagcagacaaggcccctgaacagcctagcatcttacctgatgcataggaggttcttaatcatctcccttcctctccctcatcctaacgaaaaatactagattgctgtcaaatgctatgggtatactttaaatcagtgcttgtcaaactttaatgtgcagcccaatcagctgggggaatcctgttaaaatgcaggttctgattcagtggagctggggtgagggataggaattatgcgttccttacaggcttccaggtgatgctacactgctgattggggatcattctttgagtggcaagaatttgacactactaagtttcattacttaacacaaccatcacataaaagccctcaaaaggcaccagtctaaacaataagcccttccacttcagcctcatgcaggcactgaccctgccaagtgtccagcactagagaggccaggcataatagacatatcctttggtcttgggaggatcacgacaccctcctacaggaagatcttgcaattgtttcctcacctcttctggttttttgatcttaccttttgcctctgatgataattaccctttaattaccacccaccaccttgtcatctaaataattatagtaagtgcagcctgcacctctgccagaagatctttaaacaaaatgataaaaacaagttcctaaactgccaattaaaaaaagagacaaaactgacccaaataaaacagtcatgtgcatcccacccacagtggaagacatggacttgtttttcatataaactacagaagaagttgatttattttgaaacaagaaaaagtactgattcagtatttaggaaattgtaaatgtcagaatataaattctgcagtcaggtaggcaaaacaatccaaccacactaaaatccaccttaaattcctcttgggaagagctgcagggtctctgaactatttttcctttatttggagtttccccgattataccggagggagctggataacttctgggtgcattaaaagcaaattatccatttgtgggagaagggcgggcttctcactgaaagcaattagtagttttctaatttcccaggtgggtctccattaaccgcctaacaaacaccaaggctgtcggagtccgacgaatcatgcacctctcttagggggaactggttgcgctactctttagaacgctgttttcccatggtagccttaaaaaaaacttaccaattttctgaattaggtaacacattgaatgggaaaaacctaagatagcacaaaaaggcgtacagcgaaaaattaagactccttcctcccgtcatccgccacctcactgtcctccctagaggcaatcgctggttcacttctttaaactttttattatggaaaatttcagatacacaagtaaagagactgtatgatgagcacatatgctcgcatcacgcagcttcaacgatgaaaaacgttctgccagcttgttttattcctctcccccagttttcataggcgtattttacagtcctgacaccagatcactctgtcaacacatcagtaggtcttaaaaaaaaaaaaacaaaaaaccataaccacattaccgttaccacacccaacaaagttaatgataattgctcaataccatccaatattctcggggccactttcaatcggtgaggggcagacggacttagaggaaggactgcagggctggaggggcgcgaaaaagcgaggggcgacgctgctcgtggcctcgggtgtccggcgcctcgcggtccccgccatcgtcacctacgccgggccaggaccgaccaggccaggtcgagggcggctcttgaccacgcgccccctgcctcccagctcccgggcggcggcctccgcaggcccggcacagctgcacagcccgcggtccccaggcaccggcgggtccctggaggggaagcgattgatacagctgcctgcactgcgccacccgcccggctgcccatctccgtggcacctgcgtctcccggctgggccgggagctagaagtggctgccgagaccgggagggcccggccagtcgcccgctcccgctcccgcgcctggccctcggcccgcgacctcgcggacctggactacaactcccgtggggctccgacggccgggccaatggcgggcgcccggagcatgcggggcgcagcgcctgcgcggcggtttgagtaagcggctgcgcgattggctgcggggtcgggcggccgcgcggggactgtgggaagcggagtgaCggaGCGAGCGGCTGTTGGAGGAAGGAGGTGGGGGCCGGGAGCGCAAATGGCGTTGAGATGGTTCAGGGCCCTGTTCAAACTCCAGCACTGACCATTCACCGGCGGAAGCGcDNA Sequence of the Mouse WAPL (SEQ ID NO: 30)ncggccgccagggaggcctaggccctgtccggccggcgcgcctgaggtggggagggaagttgcggggccgccgctcaccccccaccccccctgtcgcccgagcttcctatttaccgaagcggagccgcggactgtgacggcagcagagcccctcgcccctctcggtggcaccggtcggcactggtctctcgcgcggggctcccgcgcccgcccgcgggccgttgggagccggagaggcggaggcggcccgaggccaaagcacccgccaggcgccgaggggaatatgaaacaggtgtcaaaATGACATCCAGATTTGGAAAAACTTACAGTAGGAAAGGAGGAAATGGCAGTTCAAAATTTGATGAAGTTTTTTCCAACAAACGGACTACTCTTAGTACAAAATGGGGTGAGACCACATTTATGGCTAAATTAGGGCAGAAGAGGCCCAATTTCAAACCAGATATTCAAGAAATTCCGAAGAAACCTAAAGTAGAAGAAGAAGATACTGGAGATCCCTTTGGTTTTGATAGTGATGATGAGTCTCTACCTGTTTCTTCAAAAAATTTAGCCCAGGGTAAGGGTTCATCTTACTCAGAATCTAGTGAGGCTGCTCAGCTGgAAGAAGTCACTTCTGTATTTGAAGCTAATAGCAAATGTAGTCATGTGGTGGGTGAAGACAGTTTTGCTTCCGACAGATGCTTACTTGTGGAGGATACTTTAATTGGGAAAGAGAAGAGCATAaGTAGAATTCCAGAAGACAACGCAAACAAAAGTAGTTGCACTAAGTTGCTAACTTCAGATAAAGTGGAGAATTTTAGTGAAGAACATGAAAAAAATAGTCACCACTTTCACAAAAATGCTGAAGATAGTACTAAGAAACCCAATGCAGAAACCGCAGTGGCTTCTGAATATAAAGCTGATGAAACTAAAGAAACAAATGATACTTGGAACTCCCAGTCTGGAAAAAGAACAGAGTCTCCATCTGAAAGTTGTCCAGTCAAAGGATCTGTAAGAACTGGTTTATATGAATGGGATAATGATTTTGAAGATATCAGGTCAGAAGACTGTATTTTAAGTTTGGATAATGAGTCTCTTTTGGAGATGAAAGACGAGGATTTAAAAAATCGGATTGGAGGATTGGAAAATCTAAATGAAACCTTTGAAGAAGATATCATACAAAGTGTTCTTAGGCCAAGCAACTGTAGGACGTACTGTAGGGCCAATAAAGCGAGATCCTCACAGGGAGCATCAAATTTTGATAAGCTAATGGATGGCACCAGTCAGTCCTTAGCCAAAGCAAACAGTGAATCAAGTAAAGATGGCCTGAATCAGGCAAAGAAAGGTAGTGCAAGTTGTGGGACCAGTTTTCGAGGAACAGTTGGACGGACTAGAGATTACACTGTTTTACATCCATCTTGCTTGTCAGTGTGTAATGTTACCATCCAGGATACTATGGAACGGAGTATGGATGAGTTCACCGCATCCACTCCTGCAGATTTAGGAGAGGCTGGCCGGCTCAGAAAAAAGGCAGATATTGCAACCTCCAAGACCACTACTAGATTTCGACCTAGTAATACTAAATCCAAAAAGGATGTTAAACTTGAATTTTTTGGTTTTGAAGATCATGATGAGACAGGAGGTGATGAAGGGGGTTCTGGAAGTTCTAATTACAAAATTAAATATTTTGGCTTTGACGATCTCAGCGAAAGTGAAGATGATGATGATGACGACTGTCAAGTGGAAAGAAAGAAAGACAAAAAAAGAACTAAAACAGCTCCATCACCTTCCCAGCAGCCTCCTCCTGAAAGCAGCGACAATTCCCAGGATAGTCAGTCTAGTACTAATAATGCAGAAAACTTGGATTTTACAGAGGACTTGCCTGGTGTGCCTGAGAGTGTGAAGAAGCCCATAAGTAAACAAGGAGATAAATCCAAGGAAAATACCAGAAAGATTTTTAGTGGCCCCAAACGGTCACCTACAAAAGCTGTATATAATGCCAGGCATTGGAACCATCCAGACTCGGAAGAATTGCCTGGACCACCAATAGCAAAACCTCAGCGTGTCACAGTGAGGCTGTCTTCAAAGGAACCAAATCAAAAAGATGATGGAGTTTTTAAGGCTCCTGCACCACCACTCAAAGTGATAAAAACTGTGACAATACCTACTCAGCCCTACCAAGAAATAGTTACTGCACTGAAATGCAGAAAAGAAGACAAAGAATTATATACGGTTGTTCAGCACGTGAAACACTTCAATGATGTGGTGGAATTTGGTGAAAATCAAGAGTTCACTGATGACATTGAATACTTGTTAAGTGGCTTAAAGAGTACTCAGCCTCTAAACACACGTTGCCTTAGTGTTATCAGCTTAGCTACTAAATGTGCCATGCCCAGTTTTCGGATGCATCTGAGGGCACATGGGATGGTTGCAAtGGTCTTTAAAACTCTGGATGATTCCCAGCATCATCAGAATCTGTCCCTCTGTACAGCTGCTCTCATGTACATATTGAGTAGAGACCGtTTGAACATGGATCTTGATAGGGCCAGCCTAGATCTCATGATTCGGCTTGTGGAGTTGGAACAAGATGCCTCTTCAGCTAAGCTACTGAATGAAAAAGACATGAACAAGATCAAAGAAAAGATCCGAAGACTCTGTGAAACTGTGCACAACAAGCATCTTGATCTAGAAAACATAACGACTGGTCATTTAGCTATGGAGACATTGCTGTCCCTCACTTCCAAACGAGCAGGAGATTGGTTTAAAGAAGAGCTCCGACTTCTGGGTGGTCTGGATCATATTGTAGATAAAGTAAAAGAGTGTGTGGATCATTTAAGTAGAGATGATGAGGACGAAGAGAAACTAGTAGCCTCATTATGGGGAGCAGAGAGATGTTTACGAGTTTTAGAGAGTGTAACAGTGCATAATCCAGAGAATCAAAGCTACTTGATAGCCTATAAAGATTCACAACTCATTATTTCATCAGCTAAAGCATTACAGCATtGTGAAGACCTGAATCAGCAGTACAACCGTGCTGAGAACAGCATCTGTGTAGCAGACAGTAACCCTCTGCCTTACCAGAATGTAACTAACCATGtgGGcaAAGCAGTGGAGGACTGCaTGAGGGCTATAATTGGAGTATTGCTCAATTTAACTAATGATAATGAGTGGGGCAGCACAAAGACAGGAGAACAAGAAGGACTCATAGGCACAGCGATGAACTGTGTTCTTCAGGTTCCAAAGTACCTACCTCAGGAGCAGAGATTTGATATTCGAGTGCTGGGATTGGGTCTACTCATAAACCTGGTGGAGTATAGTGcCCGGAATCGACACTGCCTTGTCAACATGCAAACATCCTGTTCCTTTGATTCCTCCTTCTCTAGTGGAGAAGGCGATCATAGTTTAAGGCTAGCCGGACAAGTTCATGCTGTTCAAGCTTTAGTGCAGCTATTTCTCGAACGAGAGAGAGCAGCACAATTGGCAGAAAGTAAAACAGATGAATTGATTAAAGATGCTCCTACCACTCAGCATGATAAGAGTGGAGAGTGGCAAGAAACAAGTGGAGAAATACAGTGGGTATCAACTGAAAAGACTGATGGTGCAGAGGAGAAGCAGAAGAAGGAGGAGGAGGATGAAGAACTTGACCTCAATAAAGCCCTTCAGCATGCTGGCAAACACATGGAGGATTGCATCGTAGCCTCCTACACAGCCCTGCTTCTTGGGTGTCTCTGCCAGGAAAGTCCAATCAATGTAACTACAGTAAGGGAATATCTTCCAGAAGGAGATTTCTCCATAATGACAGAGATGCTTAAAAAGTTCTTAAGCTTCATGAATCTTACGTGTGCTGTTGGAACAACAGGCCAGAAGTCTATCTCTAGAGTGATTGAATATTTGGAACATTGCTAGctgctttacctttgcttcaggtgcttggtaatgctgaagctatccttagacaaagaaaattggatttttatgatcacccgatttcttcatcatgcattctgcgtttgctaaatgacagttactacatcaatctgcagctatcaaaaatgagggaaaaggttcaggctgttaacaatcccatgcagtatttaaatacacttac

In the description and the drawings, when abbreviating nucleotidesand/or amino acids, the abbreviations are those in accordance withIUPAC-IUB Commission on Biochemical Nomenclature or conventionalabbreviations in the art. Examples are listed below. When opticalisomers exist in an amino acid, an L-isomer is indicated unlessspecifically indicated.

DNA: deoxyribonucleic acid;

cDNA: complementary deoxyribonucleic acid;

A: adenine;

T: thymine;

G: guanine;

C: cytosine;

I: inosine;

R: adenine (A) or guanine (G);

Y: thymine (T) or cytosine (C);

M: adenine (A) or cytosine (C);

K: guanine (G) or thymine (T);

S: guanine (G) or cytosine (C);

W: adenine (A) or thymine (T);

B: guanine (G), guanine (G) or thymine (T);

D: adenine (A), guanine (G) or thymine (T);

V: adenine (A), guanine (G) or cytosine (C);

N: adenine (A), guanine (G), cytosine (C) or thymine (T) or unknown oranother base;

RNA: ribonucleic acid;

mRNA: messenger ribonucleic acid;

dATP: deoxyadenosine triphosphate;

dTTP: deoxythymidine triphosphate;

dGTP: deoxyguanosine triphosphate;

dCTP: deoxycytidine triphosphate;

ATP: adenosine triphosphate;

EDTA: ethylenediaminetetraacetic acid;

SDS: sodium dodecyl sulfate;

BHA: benzhydrylamine;

pMBHA: p-methylbenzhydrylamine;

Tos: p-toluenesulfonyl;

Bzl: benzyl;

Bom: benzyloxymethyl;

Boc: t-butyloxycarbonyl;

DCM: dichloromethane;

HOBt: 1-hydroxybenzotriazole;

DCC: N,N′-dicyclohexylcarbodiimide;

TFA: trifluoroacetic acid;

DIEA: diisopropylethylamine;

Gly or G: glycine;

Ala or A: alanine;

Val or V: valine;

Leu or L: leucine;

Ile or I: isoleucine;

Ser or S: serine;

Thr or T: threonine;

Cys or C: cysteine;

Met or M: methionine;

Glu or E: glutamic acid;

Asp or D: aspartic acid;

Lys or K: lysine;

Arg or R: arginine;

His or H: histidine;

Phe or F: phenylalanine;

Tyr or Y: tyrosine;

Trp or W: tryptophan;

Pro or P: proline;

Asn or N: asparagine;

Gln or Q: glutamine;

pGlu: pyroglutamic acid;

Tyr(I): 3-iodotyrosine;

DMF: N,N-dimethylformamide;

Fmoc: N-9-fluorenylmethoxycarbonyl;

Trt: trityl;

Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl;

Clt: 2-chlorotrityl;

Bu^(t): t-butyl; and

Met(O): methionine sulfoxide.

INDUSTRIAL APPLICABILITY

The full-length nucleotide sequence of an hWAPL oncogene according tothe present invention allows for producing a recombinant of an hWAPLoncogenic protein encoded by the hWAPL oncogene, and investigating acancerization mechanism induced by over-expression of the hWAPLoncogenic protein in a cell strain derived from any of variousepithelial cells. Furthermore, in the present invention, a promoterregion in the identified hWAPL oncogene may provide a new target instudying cancer prevention or therapy on the basis of a mechanism ofinhibition of the cancerization mechanism induced by over-expression ofthe hWAPL oncogene, by inhibiting transcription of the oncogene.Furthermore, production of a recombinant of the full-length nucleotidesequence of the hWAPL oncogene according to the present invention, thehWAPL oncogenic protein, allows for preparation of a nucleic acid probeor PCR primer for detecting mRNA expression by transcription of thehWAPL oncogene or a specific antibody for detecting a translated hWAPLoncogenic protein peptide. Thus, it allows for using various diagnosiskits which detect over-expression of the hWAPL oncogene directlyinvolved in onset of a cancer.

The invention claimed is:
 1. An isolated polynucleotide consisting ofthe nucleotide sequence encoding the polypeptide represented by SEQ. ID.No. 1, wherein the nucleotide sequence encoding the amino acid sequenceof SEQ. ID. No. 1 is the nucleotide sequence of SEQ. ID. No.2.
 2. Arecombinant expression vector comprising a polynucleotide encoding thepolypeptide of SEQ. ID. No. 1, wherein said polynucleotide encoding thepolypeptide of SEQ. ID. No. 1 is the polynucleotide of claim 1 and isoperably linked to a promoter so as to allow expression of saidpolypeptide in a human host cell.
 3. An isolated host cell containingthe recombinant expression vector of claim 2, wherein the host cell is ahuman cell-line.
 4. A process for producing a recombinant polypeptide ofSEQ ID NO:1 or its salts, comprising the steps of: culturing the hostcell of claim 3 under conditions such that the host cell produces saidpolypeptide or its salts; and collecting said recombinant polypeptide orits salts from the culture of the host cell.
 5. A process for producinga recombinant hWAPL protein of SEQ. ID. No. 1 comprising the steps of:culturing the host cell of claim 3 under conditions such that the hostcell produces said hWAPL protein; and collecting said recombinant hWAPLprotein from the culture of the host cell.
 6. A polynucleotide probeconsisting of a nucleotide sequence that is completely complementary toa region of nucleotides 2511 to 2813 of SEQ. ID. No. 2, wherein saidpolynucleotide probe has the same length as that of the region ofnucleotides 2511 to
 2813. 7. A probe hybridization kit, comprising thepolynucleotide probe of claim 6, wherein said kit is useful fordetecting an mRNA corresponding to the nucleotide sequence of SEQ. ID.No.2 or cDNA prepared by the mRNA.
 8. A primer pair, consisting of thefollowing paired primers:5′-TTGGATCCATGACATCCAGATTTGGGAAAACATACAGTAGG-3′ (SEQ ID NO: 8); and5′-TTGAATTCCTAGCAATGTTCCAAATATTCAATCACTCTAGA-3′ (SEQ ID NO: 9).
 9. Aprimer pair, consisting of the following primers:5′-GAATTCATAGGCACAGCGCTGAACTGTGTG-3′ (SEQ ID NO: 5); and5′-TTGAATTCCTAGCAATGTTCCAAATATTCA-3′ (SEQ ID NO: 6).